Method for preparing cells overexpressing exogenous genes

Abstract

The method for preparing cells overexpressing exogenous genes in the present invention specifically relates to the application of cGAS-STING signaling pathway inhibitors in culturing electroporated cells, the method for culturing electroporated cells, the method for preparing cells overexpressing exogenous genes by electroporation, and a cell culture medium supplemented with cGAS-STING signaling pathway inhibitors. By culturing the electroporated cells, especially immune effector cells, with a culture medium containing cGAS-STING signaling pathway inhibitors, the present invention can significantly reduce the mortality rate of the electroporated cells and increase the number and proportion of viable cells after cell electroporation.

Description

[0001] This application claims the priority of a Chinese patent application with the application number 202010359691.X, the application date of April 30, 2020, and the invention title of "Method for Preparing Cells Overexpressing Exogenous Genes". Technical Field

[0002] The present invention relates to the culture of electroporated cells, and specifically to a method for preparing cells overexpressing exogenous genes. Background Art

[0003] The adoptive cell therapy (ACT) using immune effector cells expressing chimeric antigen receptors (CARs), such as CAR-T cells, has the potential to permanently change the status quo of tumor treatment. Such therapies rely on efficient, stable, and safe gene transfer platforms. Transferring the synthetic gene encoding the chimeric antigen receptor into immune effector cells, such as T cells, is the first step in achieving tumor treatment. Gene transfer techniques mainly include viral methods and non-viral methods. The viral methods mainly include using retroviral vectors or lentiviral vectors to express the CAR gene, introducing the CAR gene into immune effector cells through packaged viral particles, and integrating it into the cell genome through the integration system of the retrovirus or lentivirus itself. The advantages of the viral vector system are that viral particles can effectively transduce immune effector cells, such as T cells, and integrate into the host cell genome efficiently and stably. However, the production process of the virus is costly, time-consuming, and laborious. Moreover, in order to meet the clinical safety standards, the immune effector cells modified by the viral system need to show no replication, low genotoxicity, and low immunogenicity of the virus itself, and long-term monitoring is required after being infused into the human body, which poses certain safety hazards.

[0004] Electroporation (or electropermeabilization) is already a well-established method in some areas of medicine, but its application in biotechnology has only recently emerged. By transiently applying a high electric field pulse to cells or tissues, transient pores are formed on the cell membrane surface, leading to the entry of charged molecules into the cell. Classical electroporation results in enhanced transmembrane transport and altered electrical conductivity in cells. The effects of this process on the cell membrane are related to the intensity, repetition, duration, and number of electric pulses of electroporation. Artificial bilayers, cells, or tissues can already be permeabilized by several commonly used electroporation protocols depending on their specific properties. In electroporation-based gene transfer, foreign DNA is introduced into cells by reversible electroporation, and the foreign gene is expressed in its new host cell and inherited as the cell divides. Combining electroporation with non-viral gene modification systems that can induce stable expression of transgenes, such as transposon systems, is an effective method for modifying immune effector cells in addition to viral vector systems. Transposon systems, such as the Sleeping Beauty or PiggyBac transposon systems, contain transposase-encoding sequences that encode transposases that recognize the repeated sequences on both sides and can efficiently mediate integration into the host cell genome. The combination of transposon systems and electroporation has broad prospects for therapeutic applications and has met the requirements for clinical grade (Kebriaei P, Huls H, Jena B, Munsell M, Jackson R, et al. (2012) Infusing CD19-Directed T Cells to Augment Disease Control in Patients Undergoing Autologous Hematopoietic Stem-Cell Transplantation for Advanced B-Lymphoid Malignancies. Human Gene Therapy 23:444–450), and it has high efficiency in T cells.ACT using the transposon system relies on electroporation of T cells and tumor infiltrating lymphocytes (TIL). For this purpose, there are relatively mature commercial electroporation instruments and supporting buffers (such as Lonza Nucleofactor) on the market. Recently, there have been reports of successful construction of CAR-T cells using the SB transposon system through commercial electroporation systems (Jin Z, Maiti S, Huls H, Singh H, Olivares S, et al. (2011) The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express achimeric antigen receptor. Gene Therapy 18: 849–856; Peng PD, Cohen CJ, Yang S, Hsu C, Jones S, et al. (2009) Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity. Gene Therapy 16: 1042–1049), and the ACT method using the SB transposon system has been used in clinical trials (Kebriaei P, Huls H, Jena B, Munsell M, Jackson R, et al. (2012) Infusing CD19-Directed T Cells to Augment Disease Control in Patients Undergoing Autologous Hematopoietic Stem-Cell Transplantation for Advanced B-Lymphoid Malignancies. Human Gene Therapy 23: 444–450).

[0005] Compared with the viral vector system, the electroporation combined with the transposon system has the advantages of simple operation, low immunogenicity and genotoxicity, and low safety risk, and is an important method for ACT. However, the problems it presents are also relatively obvious: it is prone to cause excessive cell death under transient high voltage, and the transfection efficiency is low, specifically related to cell types and electroporation conditions (including voltage, waveform, pulse time, and composition of electroporation buffer). Especially for immune effector cells, such as PBMC and TIL cells, the difficulty of electroporation is relatively greater. Although there are already relatively mature electroporation instruments and supporting buffer systems on the market, the problem of high mortality rate after electroporation of immune effector cells is still relatively prominent. Therefore, there is still a need for a method that can improve the survival rate of immune effector cells after electroporation. Summary of the Invention

[0006] In a first aspect of the present invention, there is provided the use of a cGAS-STING signaling pathway inhibitor in culturing electroporated cells.

[0007] In a second aspect of the present invention, there is provided a method for culturing electroporated cells, the method comprising the step of culturing the electroporated cells in a culture medium containing a cGAS-STING signaling pathway inhibitor.

[0008] In one or more embodiments, the cGAS-STING signaling pathway inhibitor includes proteins, nucleic acids, and small molecule compounds capable of inhibiting the cGAS-STING signaling pathway.

[0009] In one or more embodiments, the proteins capable of inhibiting the cGAS-STING signaling pathway include proteins that inhibit STING expression and / or binding activity or proteins that reduce or eliminate mitochondrial membrane potential, including but not limited to ULK1, ULK2, caspase3, caspase7, and caspase9.

[0010] In one or more embodiments, the nucleic acids capable of inhibiting the cGAS-STING signaling pathway include siRNA, antisense RNA, ribozymes, gene editing vectors such as CRISPR-CAS9 gene editing vectors or TALEN gene editing vectors that target the cGAS-STING signaling pathway or reduce or eliminate mitochondrial membrane potential.

[0011] In one or more embodiments, the small molecule compounds capable of inhibiting the cGAS-STING signaling pathway include small molecule compounds that inhibit STING expression and / or binding activity or reduce or eliminate mitochondrial membrane potential, and small molecule compounds that inhibit or antagonize other member molecules in the cGAS-STING signaling pathway other than STING.

[0012] In one or more embodiments, the small molecule compounds that can inhibit STING expression and / or binding activity or reduce or eliminate mitochondrial membrane potential include carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with a structural formula as shown in Formula 1, and a compound conjugated with NO as shown in Formula 2 2 linoleic acid, compound C-176 with a structural formula as shown in Formula 3, compound C-178 with a structural formula as shown in Formula 4, compound H-151 with a structural formula as shown in Formula 5, compound INHIB1X with a structural formula as shown in Formula 6, compound INHIB2 with a structural formula as shown in Formula 7, compound INHIB9 with a structural formula as shown in Formula 8, compound Compound 18 with a structural formula as shown in Formula 9, cyclic peptide astin C with a structural formula as shown in Formula 10, compound Screening Hit 1 with a structural formula as shown in Formula 11, compound Compound 13 with a structural formula as shown in Formula 12, compound C-170 with a structural formula as shown in Formula 13, and compound C-171 with a structural formula as shown in Formula 14.

[0013]

[0014]

[0015] In one or more embodiments, the small molecule compounds that can inhibit or antagonize other member molecules except STING in the cGAS-STING signaling pathway include small molecule compounds that can inhibit or antagonize cyclic GMP-AMP synthase (cGAS). Preferably, they include quinacrine with a structural formula as shown in Formula 15, hydroxychloroquine with a structural formula as shown in Formula 16, compound X6 with a structural formula as shown in Formula 17, compound PF-06928215 with a structural formula as shown in Formula 18, compound RU.365 with a structural formula as shown in Formula 19, compound RU.521 with a structural formula as shown in Formula 20, suramin with a structural formula as shown in Formula 21, compound G150 with a structural formula as shown in Formula 22, and compound VIII with a structural formula as shown in Formula 23.

[0016]

[0017]

[0018] In one or more embodiments, the small molecule compounds capable of inhibiting or antagonizing other member molecules other than STING in the cGAS-STING signaling pathway include small molecule compounds capable of inhibiting or antagonizing TANK-binding kinase 1 (TBK1). Preferably, it includes compound BX795 with the structural formula shown in Formula 24, compound Tozasertib with the structural formula shown in Formula 25, compound Tozasertib-15a with the structural formula shown in Formula 26, compound 20b with the structural formula shown in Formula 27, compound 4-azabenzimidazole hit 1a with the structural formula shown in Formula 28, compound CYT387 with the structural formula shown in Formula 29, compound Domainex with the structural formula shown in Formula 30, compound Amgen Compound II with the structural formula shown in Formula 31, compound MRT67307 with the structural formula shown in Formula 32, and compound AZ13102909 with the structural formula shown in Formula 33.

[0019]

[0020]

[0021] In one or more embodiments, the application is the application of the small molecule compounds capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential in culturing electroporated immune effector cells. Preferably, it is the application of any one or more of the small molecule compounds in Formulas 1-14 in culturing electroporated immune effector cells.

[0022] In one or more embodiments, the application is the application of the small molecule compounds capable of inhibiting or antagonizing other member molecules other than STING in the cGAS-STING signaling pathway in culturing electroporated immune effector cells.

[0023] In one or more embodiments, the application is the application of the small molecule compounds capable of inhibiting or antagonizing cyclic GMP-AMP synthase (cGAS) in culturing electroporated immune effector cells. Preferably, it is the application of any one or more of the small molecule compounds in Formulas 15-23 in culturing electroporated immune effector cells.

[0024] In one or more embodiments, the application is the application of the small molecule compounds capable of inhibiting or antagonizing TANK-binding kinase 1 (TBK1) in culturing electroporated immune effector cells. Preferably, it is the application of any one or more of the small molecule compounds in Formulas 24-33 in culturing electroporated immune effector cells.

[0025] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of the small - molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small - molecule compound capable of inhibiting or antagonizing cGAS.

[0026] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of any one or more small - molecule compounds selected from Formulas 1 - 14 and any one or more small - molecule compounds selected from Formulas 15 - 23. Preferably, the application is the co - application in cultured and electroporated immune effector cells of any one small - molecule compound selected from Formulas 1 - 14 and any one small - molecule compound selected from Formulas 15 - 23.

[0027] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of the small - molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small - molecule compound capable of inhibiting or antagonizing TBK1.

[0028] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of any one or more small - molecule compounds selected from Formulas 1 - 14 and any one or more small - molecule compounds selected from Formulas 24 - 33. Preferably, the application is the co - application in cultured and electroporated immune effector cells of any one small - molecule compound selected from Formulas 1 - 14 and any one small - molecule compound selected from Formulas 24 - 33.

[0029] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of the small - molecule compound capable of inhibiting or antagonizing cGAS and the small - molecule compound capable of inhibiting or antagonizing TBK1.

[0030] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of any one or more small - molecule compounds selected from Formulas 15 - 23 and any one or more small - molecule compounds selected from Formulas 24 - 33. Preferably, the application is the co - application in cultured and electroporated immune effector cells of any one small - molecule compound selected from Formulas 15 - 23 and any one small - molecule compound selected from Formulas 24 - 33.

[0031] In one or more embodiments, the application is the co - application in cultured and electroporated immune effector cells of the small - molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, the small - molecule compound capable of inhibiting or antagonizing cGAS, and the small - molecule compound capable of inhibiting or antagonizing TBK1.

[0032] In one or more embodiments, the application is the co - application of any one or more small - molecule compounds selected from Formula 1 - 14, any one or more small - molecule compounds selected from Formula 15 - 23, and any one or more small - molecule compounds selected from Formula 24 - 33 in culturing electro - transfected immune effector cells. Preferably, the application is the co - application of any one small - molecule compound selected from Formula 1 - 14, any one small - molecule compound selected from Formula 15 - 23, and any one small - molecule compound selected from Formula 24 - 33 in culturing electro - transfected immune effector cells.

[0033] In one or more embodiments, the method is a method for culturing electro - transfected immune effector cells, and the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing the small - molecule compound that can inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential.

[0034] In one or more embodiments, the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing any one or more small - molecule compounds selected from Formula 1 - 14.

[0035] In one or more embodiments, the method is a method for culturing electro - transfected immune effector cells, and the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing the small - molecule compound that can inhibit or antagonize cGAS.

[0036] In one or more embodiments, the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing any one or more small - molecule compounds selected from Formula 15 - 23.

[0037] In one or more embodiments, the method is a method for culturing electro - transfected immune effector cells, and the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing the small - molecule compound that can inhibit or antagonize TBK1.

[0038] In one or more embodiments, the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing any one or more small - molecule compounds selected from Formula 24 - 33.

[0039] In one or more embodiments, the method includes the step of culturing the electro - transfected immune effector cells in a culture medium containing the small - molecule compound that can inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential and the small - molecule compound that can inhibit or antagonize cGAS.

[0040] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing one or more small molecule compounds selected from any one of formulas 1-14 and one or more small molecule compounds selected from any one of formulas 15-23.

[0041] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK1.

[0042] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing one or more small molecule compounds selected from any one of formulas 15-23 and one or more small molecule compounds selected from any one of formulas 24-33.

[0043] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK1.

[0044] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing one or more small molecule compounds selected from any one of formulas 1-14 and one or more small molecule compounds selected from any one of formulas 24-33.

[0045] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK1.

[0046] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing one or more small molecule compounds selected from any one of formulas 1-14, one or more small molecule compounds selected from any one of formulas 15-23, and one or more small molecule compounds selected from any one of formulas 24-33.

[0047] In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing one small molecule compound selected from any one of formulas 1-14, one small molecule compound selected from any one of formulas 15-23, and one small molecule compound selected from any one of formulas 24-33.

[0048] In one or more embodiments, the method or application includes, after the electroporation ends, transferring the electroporated cells into a culture medium and culturing for 0.5 - 8 hours, then adding a cGAS-STING signaling pathway inhibitor into the culture medium and continuing the culture.

[0049] In one or more embodiments, the cells are immune effector cells.

[0050] In one or more embodiments, the immune effector cells are selected from one or more of T cells, TIL cells, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.

[0051] In one or more embodiments, the immune effector cells are selected from one or more of T cells, TIL cells, and CAR-T cells.

[0052] In one or more embodiments, the culture medium is a culture medium for culturing immune effector cells.

[0053] In one or more embodiments, the culture medium is a culture medium for T cells, TIL cells, or CAR-T cells.

[0054] In one or more embodiments, the culture medium is selected from CTS TM any one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, it is CTS TM serum-free cell culture medium.

[0055] In one or more embodiments, the final concentration of the cGAS-STING signaling pathway inhibitor in the culture medium is in the range of 0.02 - 100 μM, preferably in the range of 0.02 - 80 μM, more preferably in the range of 0.5 - 5 μM, and even more preferably in the range of 0.5 - 2.5 μM.

[0056] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound that can inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential, and its final concentration is in the range of 0.5 - 5 μM, preferably in the range of 0.5 - 1 μM.

[0057] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound that can inhibit or antagonize cGAS, and its final concentration is in the range of 0.5 - 5 μM, preferably in the range of 2.5 - 5 μM.

[0058] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting or antagonizing TBK1, and its final concentration ranges from 0.02 to 10 μM, preferably from 0.02 to 1 μM.

[0059] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing cGAS, and its final concentration ranges from 0.02 to 10 μM, preferably from 0.02 to 5 μM.

[0060] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK1, and its final concentration ranges from 0.02 to 10 μM, preferably from 0.02 to 5 μM.

[0061] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK1, and its final concentration ranges from 0.02 to 10 μM, preferably from 0.02 to 5 μM.

[0062] In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK1, and its final concentration ranges from 0.02 to 20 μM, preferably from 0.02 to 5 μM.

[0063] The present invention also provides a method for preparing cells overexpressing an exogenous gene by electroporation, and the method includes:

[0064] 1) Introducing a nucleic acid containing an exogenous gene to be expressed into cells by electroporation;

[0065] 2) Culturing the cells electroporated with the exogenous gene in step 1) with a culture medium containing a cGAS-STING signaling pathway inhibitor;

[0066] Among them, the electrotransformed cells are cultured for 0 - 8 h, preferably 0 - 5 h, more preferably 0 - 3 h, and even more preferably 0 - 1 h, and then the cGAS-STING signaling pathway inhibitor is added to the medium; preferably, after adding the cGAS-STING signaling pathway inhibitor, its final concentration in the medium is in the range of 0.02 - 100 μM, preferably 0.02 - 80 μM; more preferably, in the range of 0.5 - 5 μM; even more preferably, in the range of 0.5 - 1 μM or in the range of 2.5 - 5 μM.

[0067] Preferably, the cGAS-STING signaling pathway inhibitor includes any one or more of small molecule compounds that can inhibit STING expression and / or binding activity or reduce or eliminate mitochondrial membrane potential, small molecule compounds that can inhibit or antagonize cGAS, and small molecule compounds that can inhibit or antagonize TBK1; preferably, the cells are immune effector cells.

[0068] In one or more embodiments, the small molecule compounds that can inhibit STING expression and / or binding activity or reduce or eliminate mitochondrial membrane potential include any one or more small molecule compounds selected from Formulas 1 - 14.

[0069] In one or more embodiments, the small molecule compounds that can inhibit or antagonize cGAS include any one or more small molecule compounds selected from Formulas 15 - 23.

[0070] In one or more embodiments, the small molecule compounds that can inhibit or antagonize TBK1 include any one or more small molecule compounds selected from Formulas 24 - 33.

[0071] The present invention also provides a cell culture medium containing a cGAS-STING signaling pathway inhibitor.

[0072] In one or more embodiments, the cell culture medium is a medium for culturing immune effector cells.

[0073] In one or more embodiments, the medium for culturing immune effector cells is a medium for T cells, TIL cells or CAR-T cells.

[0074] In one or more embodiments, the medium for culturing immune effector cells is selected from CTS TM any one of serum-free cell culture medium, DMEM medium and RPMI1640 medium; preferably, it is CTS TM serum-free cell culture medium.

[0075] In one or more embodiments, the cGAS-STING signaling pathway inhibitor includes any one or more of small molecule compounds capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, small molecule compounds capable of inhibiting or antagonizing cGAS, and small molecule compounds capable of inhibiting or antagonizing TBK1.

[0076] In one or more embodiments, the small molecule compounds capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential include any one or more small molecule compounds selected from Formulas 1-14.

[0077] In one or more embodiments, the small molecule compounds capable of inhibiting or antagonizing cGAS include any one or more small molecule compounds selected from Formulas 15-23.

[0078] In one or more embodiments, the small molecule compounds capable of inhibiting or antagonizing TBK1 include any one or more small molecule compounds selected from Formulas 24-33. Description of the Drawings

[0079] Figure 1 : Bright-field and fluorescence field images of TIL cells cultured for 5 days after adding G150 at 0 h after electroporation of pNB328-EGFP plasmid;

[0080] Figure 2 : Flow cytometry analysis results of EGFP-positive cells in TIL cells after adding G150 to 2.5 μM at 0 h, 1 h, and 5 h after electroporation of pNB328-EGFP plasmid and culturing for 5 days;

[0081] Figure 3 : Number of live cells in the treatment group with G150 added to 2.5 μM at 0 h, 1 h, and 5 h after electroporation of pNB328-EGFP plasmid into TIL cells and the control group without G150 added, after culturing for 5 days;

[0082] Figure 4 : Proportion of live cells in the treatment group with G150 added to 2.5 μM at 0 h, 1 h, and 5 h after electroporation of pNB328-EGFP plasmid into TIL cells and the control group without G150 added, after culturing for 5 days;

[0083] Figure 5 : Proportion of EGFP-positive cells in the treatment group with G150 added to 2.5 μM at 0 h, 1 h, and 5 h after electroporation of pNB328-EGFP plasmid into TIL cells and the control group without G150 added, after culturing for 5 days;

[0084] Figure 6: The number of viable cells in the treatment group with G150 added to 5 μM at 0 h, 1 h, and 5 h after activating T cells transfected with pNB328-EGFP plasmid and the control group without G150 added after culturing for 5 days;

[0085] Figure 7 : The proportion of viable cells in the treatment group with G150 added to 5 μM at 0 h, 1 h, and 5 h after activating T cells transfected with pNB328-EGFP plasmid and the control group without G150 added after culturing for 5 days;

[0086] Figure 8 : The proportion of EGFP-positive cells in the treatment group with G150 added to 5 μM at 0 h, 1 h, and 5 h after activating T cells transfected with pNB328-EGFP plasmid and the control group without G150 added after culturing for 5 days;

[0087] Figure 9 : The number of viable cells in the treatment group with H-151 added to 0.5 μM at 0 h, 1 h, and 5 h after TIL transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0088] Figure 10 : The proportion of viable cells in the treatment group with H-151 added to 0.5 μM at 0 h, 1 h, and 5 h after TIL transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0089] Figure 11 : The proportion of EGFP-positive cells in the treatment group with H-151 added to 0.5 μM at 0 h, 1 h, and 5 h after TIL transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0090] Figure 12 : The number of viable cells in the treatment group with H-151 added to 1 μM at 0 h, 1 h, and 5 h after activating T transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0091] Figure 13 : The proportion of viable cells in the treatment group with H-151 added to 1 μM at 0 h, 1 h, and 5 h after activating T transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0092] Figure 14 : The proportion of EGFP-positive cells in the treatment group with H-151 added to 1 μM at 0 h, 1 h, and 5 h after activating T transfected with pNB328-EGFP plasmid and the control group without H-151 added after culturing for 5 days;

[0093] Figure 15: The number of viable cells in the treatment group with IRAK-IN-4 added to 2 μM at 0 h, 1 h, and 5 h after electroporating TIL cells with the pNB328-EGFP plasmid and the control group without IRAK-IN-4 added after culturing for 5 days;

[0094] Figure 16 : The proportion of viable cells in the treatment group with IRAK-IN-4 added to 2 μM at 0 h, 1 h, and 5 h after electroporating TIL cells with the pNB328-EGFP plasmid and the control group without IRAK-IN-4 added after culturing for 5 days;

[0095] Figure 17 : The proportion of EGFP-positive cells in the treatment group with IRAK-IN-4 added to 2 μM at 0 h, 1 h, and 5 h after electroporating TIL cells with the pNB328-EGFP plasmid and the control group without IRAK-IN-4 added after culturing for 5 days;

[0096] Figure 18 : The number of viable cells in the treatment group with C-170 added to 2 μM at 0 h, 1 h, and 5 h after electroporating activated T cells with the pNB328-EGFP plasmid and the control group without C-170 added after culturing for 5 days;

[0097] Figure 19 : The proportion of viable cells in the treatment group with C-170 added to 2 μM at 0 h, 1 h, and 5 h after electroporating activated T cells with the pNB328-EGFP plasmid and the control group without C-170 added after culturing for 5 days;

[0098] Figure 20 : The proportion of EGFP-positive cells in the treatment group with C-170 added to 2 μM at 0 h, 1 h, and 5 h after electroporating activated T cells with the pNB328-EGFP plasmid and the control group without C-170 added after culturing for 5 days. Detailed implementation manners

[0099] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form preferred technical solutions.

[0100] Stimulator of interferon genes (STING) is an important molecule in the innate immune response and plays an important role in defending against viral and intracellular bacterial infections and mediating the production of type I interferons. As an important part of the signal transduction cascade reaction, STING can not only play an immune defense role during pathogen (virus, bacteria, parasite, etc.) infections but also play an anti-tumor immune response during tumorigenesis. When the cGAS-STING signaling pathway is overactivated, it may also cause a series of autoimmune diseases.

[0101] The present invention discovers that after cells, especially immune effector cells, are transferred into a culture medium for culturing for a period of time after the end of electroporation, adding a cGAS-STING signaling pathway inhibitor for culturing can significantly improve the survival rate of the cells after electroporation, thereby completing the present invention.

[0102] In this article, the cells can be any cells of interest, especially cells conventionally used for electroporation in the art to express foreign genes, and can be eukaryotic cells (such as animal cells and plant cells) and prokaryotic cells (such as Escherichia coli and other bacteria, etc.). For example, the cells can be human cells. Examples of cells include but are not limited to HEK293 cells, MDCK cells, and Hela cells, etc. In certain embodiments, the cells are immune cells. In this article, immune effector cells refer to immune cells that participate in clearing foreign antigens and exercising effector functions in the immune response, including but not limited to one or more of T cells, TIL cells, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Preferably, in certain embodiments, the immune effector cells applicable to the method of the present invention are selected from one or more of T cells, TIL, and CAR-T cells.

[0103] In this article, the cGAS-STING signaling pathway inhibitor can be various preparations well-known in the art that can inhibit the cGAS-STING signaling pathway, including but not limited to proteins, nucleic acids, and small molecule compounds that can inhibit the cGAS-STING signaling pathway. For example, the proteins that can inhibit the cGAS-STING signaling pathway include proteins that inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential, including but not limited to ULK1, ULK2, caspase3, caspase7, and caspase9. The nucleic acids that can inhibit the cGAS-STING signaling pathway include but are not limited to siRNA, antisense RNA, ribozymes, and gene editing vectors that target the cGAS-STING signaling pathway or reduce or eliminate the mitochondrial membrane potential, such as the CRIPR-CAS9 gene editing vector or the TALEN gene editing vector. The small molecule compounds that can inhibit the cGAS-STING signaling pathway include but are not limited to small molecule compounds that can inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential, small molecule compounds that can inhibit or antagonize cGAS, and small molecule compounds that can inhibit or antagonize TBK1. The small molecule compounds that can inhibit STING expression and / or binding activity or reduce or eliminate the mitochondrial membrane potential include but are not limited to the small molecule compounds in Formulas 1-14. The small molecule compounds that can inhibit or antagonize cGAS include but are not limited to the small molecule compounds in Formulas 15-23. The small molecule compounds that can inhibit or antagonize TBK1 include but are not limited to the small molecule compounds in Formulas 24-33.

[0104] In certain embodiments, the present invention relates to culturing electroporated cells using small molecule compounds capable of inhibiting STING expression and / or binding activity or small molecule compounds that reduce or eliminate mitochondrial membrane potential, for example, culturing electroporated cells using any one or more of the small molecule compounds of Formulas 1-14.

[0105] In certain embodiments, the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 is used to culture electroporated cells. In certain embodiments, the compound H-151 of Formula 5 is used to culture electroporated cells. In certain embodiments, the small molecule compound Compound 18 of Formula 9 is used to culture electroporated cells. In certain embodiments, Compound 13 of Formula 12 is used to culture electroporated cells. In certain embodiments, the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound H-151 of Formula 5 are used to culture electroporated cells. In certain embodiments, the compound H-151 of Formula 5 and the small molecule compound 18 of Formula 9 are used together to culture electroporated cells. In certain embodiments, the compound H-151 of Formula 5 and Compound 13 of Formula 12 are used to culture electroporated cells.

[0106] In certain embodiments, the present invention relates to culturing electroporated cells using small molecule compounds capable of inhibiting or antagonizing cGAS, for example, culturing electroporated cells using any one or more of the small molecule compounds of Formulas 15-23.

[0107] In certain embodiments, the compound quinacrine of Formula 15 is used to culture electroporated cells. In certain embodiments, the compound hydroxychloroquine of Formula 16 is used to culture electroporated cells. In certain embodiments, the compound RU.365 of Formula 19 is used to culture electroporated cells. In certain embodiments, the compound RU.521 of Formula 20 is used to culture electroporated cells. In certain embodiments, the compound quinacrine of Formula 15 and the compound hydroxychloroquine of Formula 16 are used together to culture electroporated cells. In certain embodiments, the compound quinacrine of Formula 15 and the compound RU.365 of Formula 19 are used together to culture electroporated cells. In certain embodiments, the compound quinacrine of Formula 15 and the compound RU.521 of Formula 20 are used together to culture electroporated cells.

[0108] In certain embodiments, the present invention relates to culturing electroporated cells using small molecule compounds capable of inhibiting or antagonizing TBK1, for example, culturing electroporated cells using any one or more of the small molecule compounds of Formulas 24-33.

[0109] In certain embodiments, electroporated cells are cultured using a compound BX795 of formula 24. In certain embodiments, electroporated cells are cultured using a compound Tozasertib of formula 25. In certain embodiments, electroporated cells are cultured using a compound 20b of formula 27. In certain embodiments, electroporated cells are cultured using a compound CYT387 of formula 29. In certain embodiments, electroporated cells are cultured using a compound BX795 of formula 24 and a compound Tozasertib of formula 25 together. In certain embodiments, electroporated cells are cultured using a compound BX795 of formula 24 and a compound 20b of formula 27 together. In certain embodiments, electroporated cells are cultured using a compound 20b of formula 27 and a compound CYT387 of formula 29 together.

[0110] In certain embodiments, the present invention relates to culturing electroporated cells using a small molecule compound capable of inhibiting STING expression and / or binding activity or a small molecule compound that reduces or eliminates mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing cGAS together, for example, culturing electroporated cells using any one of the small molecule compounds of formulas 1-14 and any one of the small molecule compounds of formulas 15-23 together.

[0111] In certain embodiments, electroporated cells are co-cultured using a compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of formula 1 and a compound quinacrine of formula 15. In certain embodiments, electroporated cells are co-cultured using a compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of formula 1 and a compound quinacrine of formula 15. In certain embodiments, electroporated cells are co-cultured using a compound H-151 of formula 5 and a compound quinacrine of formula 15. In certain embodiments, electroporated cells are co-cultured using a small molecule compound Compound 18 of formula 9 and a compound quinacrine of formula 15. In certain embodiments, electroporated cells are co-cultured using a small molecule compound Compound 13 of formula 12 and a compound quinacrine of formula 15.

[0112] In certain embodiments, the electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound hydroxychloroquine of Formula 16. In certain embodiments, the electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound hydroxychloroquine of Formula 16. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound hydroxychloroquine of Formula 16. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound hydroxychloroquine of Formula 16.

[0113] In certain embodiments, the electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound RU.365 of Formula 19. In certain embodiments, the electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound RU.365 of Formula 19. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound RU.365 of Formula 19. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound RU.365 of Formula 19.

[0114] In certain embodiments, the electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound RU.521 of Formula 20. In certain embodiments, the electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound RU.521 of Formula 20. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound RU.521 of Formula 20. In certain embodiments, the electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound RU.521 of Formula 20.

[0115] In certain embodiments, the present invention relates to co-culturing electroporated cells with a small molecule compound capable of inhibiting STING expression and / or binding activity or a small molecule compound that reduces or eliminates the mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing TBK1, for example, co-culturing electroporated cells with any one of the small molecule compounds of Formulas 1-14 and any one of the small molecule compounds of Formulas 24-33.

[0116] In certain embodiments, electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound BX795 of Formula 24. In certain embodiments, electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound BX795 of Formula 24. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound BX795 of Formula 24. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound BX795 of Formula 24.

[0117] In certain embodiments, electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound Tozasertib of Formula 25. In certain embodiments, electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound Tozasertib of Formula 25. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound Tozasertib of Formula 25. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound Tozasertib of Formula 25.

[0118] In certain embodiments, electroporated cells are co-cultured with the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound 20b of Formula 27. In certain embodiments, electroporated cells are co-cultured with the compound H-151 of Formula 5 and the compound 20b of Formula 27. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 18 of Formula 9 and the compound 20b of Formula 27. In certain embodiments, electroporated cells are co-cultured with the small molecule compound Compound 13 of Formula 12 and the compound 20b of Formula 27.

[0119] In certain embodiments, the electroporated cells are co-cultured using the compound CCCP (carbonyl cyanide 3-chlorophenylhydrazone) of Formula 1 and the compound CYT387 of Formula 29. In certain embodiments, the electroporated cells are co-cultured using the compound H-151 of Formula 5 and the compound CYT387 of Formula 29. In certain embodiments, the electroporated cells are co-cultured using the small molecule compound Compound 18 of Formula 9 and the compound CYT387 of Formula 29. In certain embodiments, the electroporated cells are co-cultured using the small molecule compound Compound 13 of Formula 12 and the compound CYT387 of Formula 29.

[0120] In certain embodiments, the present invention relates to co-culturing electroporated cells using a small molecule compound capable of inhibiting or antagonizing cGAS and a small molecule compound capable of inhibiting or antagonizing TBK1, for example, co-culturing electroporated cells using any one of the small molecule compounds of Formulas 15-23 and any one of the small molecule compounds of Formulas 24-33.

[0121] In certain embodiments, the electroporated cells are co-cultured using the compound quinacrine of Formula 15 and the compound BX795 of Formula 24. In certain embodiments, the electroporated cells are co-cultured using the compound hydroxychloroquine of Formula 16 and the compound BX795 of Formula 24. In certain embodiments, the electroporated cells are co-cultured using the compound RU.365 of Formula 19 and the compound BX795 of Formula 24. In certain embodiments, the electroporated cells are co-cultured using the compound RU.521 of Formula 20 and the compound BX795 of Formula 24.

[0122] In certain embodiments, the electroporated cells are co-cultured using the compound quinacrine of Formula 15 and the compound Tozasertib of Formula 25. In certain embodiments, the electroporated cells are co-cultured using the compound hydroxychloroquine of Formula 16 and the compound Tozasertib of Formula 25. In certain embodiments, the electroporated cells are co-cultured using the compound RU.365 of Formula 19 and the compound Tozasertib of Formula 25. In certain embodiments, the electroporated cells are co-cultured using the compound RU.521 of Formula 20 and the compound Tozasertib of Formula 25.

[0123] In certain embodiments, the electroporated cells are co-cultured using the compound quinacrine represented by Formula 15 and the compound 20b represented by Formula 27. In certain embodiments, the electroporated cells are co-cultured using the compound hydroxychloroquine represented by Formula 16 and the compound 20b represented by Formula 27. In certain embodiments, the electroporated cells are co-cultured using the compound RU.365 represented by Formula 19 and the compound 20b represented by Formula 27. In certain embodiments, the electroporated cells are co-cultured using the compound RU.521 represented by Formula 20 and the compound 20b represented by Formula 27.

[0124] In certain embodiments, the electroporated cells are co-cultured using the compound quinacrine represented by Formula 15 and the compound CYT387 represented by Formula 29. In certain embodiments, the electroporated cells are co-cultured using the compound hydroxychloroquine represented by Formula 16 and the compound CYT387 represented by Formula 29. In certain embodiments, the electroporated cells are co-cultured using the compound RU.365 represented by Formula 19 and the compound CYT387 represented by Formula 29. In certain embodiments, the electroporated cells are co-cultured using the compound RU.521 represented by Formula 20 and the compound CYT387 represented by Formula 29.

[0125] In certain embodiments, the present invention relates to co-culturing electroporated cells using small molecule compounds capable of inhibiting STING expression and / or binding activity or small molecule compounds that reduce or eliminate mitochondrial membrane potential, small molecule compounds capable of inhibiting or antagonizing TBK1, and small molecule compounds capable of inhibiting or antagonizing cGAS, for example, co-culturing electroporated cells using any one or more of the small molecule compounds in Formulas 1-14, any one or more of the small molecule compounds in Formulas 15-23, and any one or more of the small molecule compounds in Formulas 24-33. Preferably, electroporated cells are co-cultured using any one of the small molecule compounds in Formulas 1-14, any one of the small molecule compounds in Formulas 15-23, and any one of the small molecule compounds in Formulas 24-33.

[0126] Electroporation, also known as electropermeabilization, is used to introduce DNA of interest into host cells. The present invention can be implemented using various electroporation methods and electroporation reagents well known in the art. For example, LONZA I Device and the electroporation reagents provided thereby can be used. The electroporation solution can be prepared first according to the instructions of the commercially available electroporation reagent, and the electroporation plasmid is added. During electroporation, the cells are resuspended with the electroporation solution containing the electroporation plasmid, and electroporation is performed in an electroporation instrument.

[0127] The electroporation plasmid can be any plasmid of interest. The amount of the electroporation plasmid can be a conventional amount in the art. For example, electroporation is performed using 3-8 μg of the plasmid per 5×10 6 cells.

[0128] After the electroporation is completed, take out the cell suspension, add pre-warmed cell culture medium, and then culture under conventional conditions (such as 37 °C, 5% CO 2 ) for at least 0.5 hours. Then, add the cGAS-STING signaling pathway inhibitor to the cell culture medium containing the electroporated cells. Adding the cGAS-STING signaling pathway inhibitor too early or too late may not be able to increase the total number and survival rate of the electroporated cells. Therefore, preferably, the cGAS-STING signaling pathway inhibitor is added to the culture medium within 8 hours, more preferably within 5 hours, and even more preferably within 3 hours after culturing the electroporated cells. For example, in some particularly preferred embodiments, the cGAS-STING signaling pathway inhibitor is added when the electroporated cells are cultured for 0.5 - 3 h, preferably when cultured for 45 min to 2 h, and even more preferably when cultured for 1 - 2 hours.

[0129] Generally, the final concentration of the cGAS-STING signaling pathway inhibitor in the culture medium after addition is in the range of 0.02 - 100 μM, preferably in the range of 0.02 - 80 μM, more preferably in the range of 0.5 - 5 μM; even more preferably in the range of 0.5 - 1 μM or in the range of 2.5 - 5 μM.

[0130] In this article, the cell culture medium can be various media suitable for culturing cells (especially immune cells), especially the media conventionally used in the art for culturing various electroporated cells. In some embodiments, the medium is a medium for culturing immune cells, including but not limited to media for culturing one or more of T cells, TIL cells, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. In some embodiments, the medium is a medium for T cells, TIL, and / or CAR-T cells, especially for culturing electroporated T cells, TIL, and / or CAR-T cells. Exemplary media include but are not limited to CTS TM any one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, it is CTS TM serum-free cell culture medium.

[0131] After culturing the electroporated cells according to the conventional culture process, compared with the conventional electroporation method in the art, culturing with the addition of the cGAS-STING signaling pathway inhibitor can significantly increase the total number of cells and / or the survival rate of the electroporated cells.

[0132] Therefore, the present invention provides a method for culturing electroporated cells (especially immune effector cells), the method comprising, after the end of electroporation, transferring the electroporated cells into a culture medium, adding a cGAS-STING signaling pathway inhibitor to the culture medium when culturing for 0 - 8 h, and continuing the culture. Preferably, the cGAS-STING signaling pathway inhibitor is added for culture when culturing for 0 - 5 h, 0 - 3 h, 0 - 1 h or 1 - 2 h in the culture medium.

[0133] In certain embodiments, the present invention provides a method for preparing cells (especially immune effector cells) expressing an exogenous gene by electroporation, the method comprising:

[0134] 1) Introducing a nucleic acid containing an exogenous gene to be expressed into cells by electroporation;

[0135] 2) Culturing the cells electroporated with the exogenous gene in step 1) with a culture medium containing a cGAS-STING signaling pathway inhibitor;

[0136] wherein, the cGAS-STING signaling pathway inhibitor is added when culturing the electroporated cells for 0.5 - 8 h, preferably 0.5 - 5 h, more preferably 0.5 - 3 h, more preferably 0.5 - 2 h, and more preferably 1 - 2 h after the end of electroporation.

[0137] The present invention also provides the use of a cGAS-STING signaling pathway inhibitor in culturing electroporated cells (especially immune effector cells).

[0138] The present invention further provides a cell culture medium containing a cGAS-STING signaling pathway inhibitor. Preferably, the cell culture medium is a culture medium for immune effector cells. More preferably, the cGAS-STING signaling pathway inhibitor is selected from one or more of small molecule compounds capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, small molecule compounds capable of inhibiting or antagonizing cGAS, and small molecule compounds capable of inhibiting or antagonizing TBK1.

[0139] Therefore, in certain embodiments, the cell culture medium of the present invention is a culture medium for culturing immune effector cells containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential; preferably, it is a culture medium containing one or more of the small molecule compounds selected from Formula 1 - 14.

[0140] In certain embodiments, the cell culture medium of the present invention is a culture medium for culturing immune effector cells containing the small molecule compound capable of inhibiting or antagonizing cGAS; preferably, it is a culture medium containing one or more of the small molecule compounds selected from Formula 15 - 23.

[0141] In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells containing the small molecule compound capable of inhibiting or antagonizing TBK1; preferably, it is a medium containing any one or more small molecule compounds selected from Formulas 24-33.

[0142] In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing cGAS; preferably, it is a medium containing any one or more small molecule compounds selected from Formulas 1-14 and any one or more small molecule compounds selected from Formulas 15-23.

[0143] In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK1; preferably, it is a medium containing any one or more small molecule compounds selected from Formulas 1-14 and any one or more small molecule compounds selected from Formulas 24-33.

[0144] In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells containing the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK1; preferably, it is a medium containing any one or more small molecule compounds selected from Formulas 15-23 and any one or more small molecule compounds selected from Formulas 24-33.

[0145] In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells containing the small molecule compound capable of inhibiting STING expression and / or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK1; preferably, it is a medium containing any one or more small molecule compounds selected from Formulas 1-14, any one or more small molecule compounds selected from Formulas 15-23, and any one or more small molecule compounds selected from Formulas 24-33.

[0146] Conventionally used for culturing one or more of T cells, TIL cells, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages, especially a medium conventionally used for culturing T cells, TIL cells, and / or CAR-T cells. More preferably, the medium is a medium conventionally used for culturing electroporated immune effector cells. Adding an inhibitor of the cGAS-STING signaling pathway to such a medium for culturing electroporated immune effector cells can significantly increase the total number and survival rate of cells. Preferably, the final concentration of the cGAS-STING signaling pathway inhibitor in the medium is in the range of 0.02 - 100 μM, preferably in the range of 0.02 - 80 μM, more preferably in the range of 0.5 - 5 μM; even more preferably, in the range of 0.5 - 1 μM or in the range of 2.5 - 5 μM. An exemplary immune effector cell medium of the present invention is a CTS TM serum-free cell medium, DMEM medium, or RPMI1640 medium; preferably, it is a CTS TM serum-free cell medium; more preferably, it is a CTS TM serum-free cell medium containing the cGAS-STING signaling pathway inhibitor at the above-mentioned concentration.

[0147] The beneficial effect of the present invention is that by using a medium containing an inhibitor of the cGAS-STING signaling pathway, such as a small molecule compound that can inhibit STING expression and / or binding activity, or reduce or eliminate mitochondrial membrane potential, one or more of the small molecule compounds that can inhibit or antagonize cGAS, and the small molecule compounds that can inhibit or antagonize TBK1, to culture the electroporated cells, especially immune effector cells, the mortality rate of the electroporated cells can be significantly reduced, and the number and proportion of live cells after cell electroporation can be increased.

[0148] The present invention will be illustrated below by specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The electroporator used in the examples was the LONZA Nucleofector TM 2b purchased from Lonza; other methods and reagents used in the examples were conventional methods and reagents in the art.

[0149] The structural formulas of the compounds used in this application are shown below:

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] pNB328-EGFP used in the following examples was described in Chinese Patent CN105154473B, and all its contents are incorporated herein by reference.

[0156] The small molecule compounds G150, H-151 and IRAK-IN-4 used in the following examples were all purchased from MCE, and the product numbers were HY-128583, HY-112693 and HY-114181 respectively. C-170 was purchased from Cayman Chemical, and the product number was 30157. Other reagents used can be obtained through commercial channels.

[0157] Example 1: Isolation and culture of TIL cells derived from human liver cancer tissue

[0158] Collect the newly resected liver cancer specimens and immediately process them under sterile conditions. The specific method is as follows: Remove the normal tissues and necrotic areas around the liver cancer specimens, and take small tissue pieces with a size of 1-2 mm from different areas of the specimens, and place one piece in each well of a 24-well plate. Add 2 mL of complete medium (AIM-V medium containing 10% FBS) and 3000 IU / mL of IL-2 to each well. Place the 24-well plate in an incubator at 37°C and 5% CO 3 for culture. On the 5th to 6th day after the start of culture, perform a half-volume medium change for all wells. After that, according to the growth of TIL, perform a half-volume medium change every 1-2 days. Once the TIL in the wells is confluent and all adherent cells have been removed, collect the TIL in each confluent well. 2

[0159] Example 2: Preparation of human activated T cells

[0160] Coat a six-well plate with a coating solution containing 5 μg / ml anti-CD3 antibody and 5 μg / ml anti-CD28 antibody at room temperature for 2 - 4 hours. After aspirating the coating solution, wash the well plate 1 - 3 times with normal saline, and add AIM-V medium containing 2% FBS for later use; Resuscitate human peripheral blood PBMC (purchased from ALLCELLS) in a 37°C water bath. Incubate the PBMC adherently for 2 - 4 h. The non-adherent suspended cells are the naive T cells. Collect the suspended cells into a 15 ml centrifuge tube, centrifuge at 1200 rmp for 3 min, discard the supernatant, add normal saline, centrifuge at 1200 rmp for 3 min, discard the normal saline, and repeat this step; Then transfer the washed naive T cells to the antibody-coated wells containing the medium for later use, and incubate at 37°C, 5% CO 2 Perform subsequent experiments after culturing for 3 - 4 days.

[0161] Application of cGAS small molecule inhibitor G150 in the preparation of TIL cells overexpressing EGFP by electroporation in Example 3

[0162] Electroporate TIL expressing EGFP according to the following steps:

[0163] 1) Pre-add AIM-V medium to 4 wells in a 12-well plate, 2 mL per well, and then transfer it to a cell culture incubator at 37°C, 5% CO 2 Preheat for 1 hour;

[0164] 2) Prepare the electroporation solution ratio for each well's single use according to the following table:

[0165] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of the solution]]> 82 Electroporation supplement solution 18

[0166] 3) Take the TIL obtained in Example 1 into 4 EP tubes, add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL normal saline, and repeat the centrifugation step to wash the cell pellet;

[0167] 4) Add 5 μg of plasmid pNB328-EGFP to the electroporation solution prepared in 2), and then let it stand at room temperature for within 30 min;

[0168] 5) Resuspend the 4 tubes of TIL with the plasmid-containing electroporation solution prepared in 4), 100 μL per tube. Carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette. Place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation slot, start the electroporation program, and select T-020 for the electroporation program;

[0169] 6) After the electroporation is completed, carefully take out the electroporation cup, aspirate the cell suspension and transfer it to an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube, and then transfer it to the wells containing pre-warmed AIM-V medium in the 12-well plate in 1). Incubate at 37 °C and 5% CO 2 culture;

[0170] 7) Add the compound G150 shown in Formula 22 to a final concentration of 2.5 μM to 3 wells after 0 h, 1 h, and 5 h of culture respectively; do not add G150 to the remaining well as a control well, and then continue the culture.

[0171] Repeat the culture operation 3 times, and use a cell counter to detect the cell number and cell viability in each well after 5 days of culture, and take the average value.

[0172] The results are as Figures 1-5 shown. Figure 1 Show the EGFP-positive cells observed by fluorescence microscopy in TIL cells cultured for 5 days after adding G150 at 0 h after electroporation. Figure 2 Show the flow cytometry analysis results of EGFP-positive cells in TIL cells cultured for 5 days after adding G150 to 2.5 μM at 0 h, 1 h, and 5 h after electroporation. Figures 3-5 Respectively show the number of live cells, the proportion of live cells, and the proportion of EGFP-positive cells in the treatment group with G150 added to 2.5 μM at 0 h, 1 h, and 5 h after TIL electroporation of pNB328-EGFP and the control group without G150 added after 5 days of culture. The results show that adding the G150 molecule at 0 h, 1 h, and 5 h after TIL electroporation of the plasmid expressing EGFP can significantly increase the number of live cells, the proportion of live cells, and the proportion of cells with positive EGFP expression after TIL cell electroporation. This indicates that adding the cGAS inhibitor G150 can significantly improve the survival level of TIL cells after electroporation and increase the transfection efficiency of foreign genes.

[0173] Example 4 Application of cGAS small molecule inhibitor G150 in the preparation of T cells overexpressing EGFP by electroporation

[0174] Electroporate T cells expressing EGFP according to the following steps:

[0175] 1) Pre-add AIM-V medium to 4 wells in a 12-well plate, 2 mL per well, and then transfer it to a cell culture incubator at 37 °C and 5% CO 2 preheat for 1 hour;

[0176] 2) Prepare the electroporation solution ratio for each well according to the following table:

[0177] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of the solution]]> 82 Electroporation supplement solution 18

[0178] 3) Take the activated T cells obtained in Example 2 into 4 EP tubes, and add 5×106 One cell was centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and then the cell pellet was resuspended in 500 μL of normal saline. The centrifugation step was repeated to wash the cell pellet.

[0179] 4) Add 5 μg of plasmid pNB328-EGFP to the electroporation solution prepared in 2), and then let it stand at room temperature for within 30 min.

[0180] 5) Resuspend 4 tubes of T cells with the plasmid-containing electroporation solution prepared in 4), 100 μL per tube. Carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette. Place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation chamber, and start the electroporation program. Select T-020 for the electroporation program.

[0181] 6) After electroporation, carefully take out the electroporation cuvette, aspirate the cell suspension and transfer it into an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube, and then transfer it into the wells containing pre-warmed AIM-V medium in the 12-well plate in 1). Incubate at 37 °C and 5% CO 2 culture.

[0182] 7) Add compound G150 shown in formula 22 to a final concentration of 5 μM to 3 wells at 0 h, 1 h, and 5 h after culture respectively; do not add G150 to the remaining well as a control well, and then continue the culture.

[0183] Repeat the culture operation 3 times. Detect the cell number and cell viability in each well after 5 days of culture using a cell counter, and take the average value.

[0184] The results are as Figures 6-8 shown. Figures 6-8 Respectively show the number of live cells, the proportion of live cells, and the proportion of EGFP-positive cells in the treatment group with G150 added to 5 μM at 0 h, 1 h, and 5 h after electroporation of activated T cells with pNB328-EGFP and the control group without G150 added after 5 days of culture. The results show that adding G150 molecules at 0 h, 1 h, and 5 h after electroporation of T cells expressing EGFP plasmid can significantly increase the number of live cells, the proportion of live cells, and the proportion of cells positive for EGFP expression after electroporation of activated T cells. This indicates that adding the cGAS inhibitor G150 can significantly improve the survival level of activated T cells after electroporation and increase the transfection efficiency of foreign genes.

[0185] Example 5 Application of STING small molecule inhibitor H-151 in electroporation to prepare TIL cells overexpressing EGFP

[0186] Electroporate TIL cells expressing EGFP according to the following steps:

[0187] 1) Pre-add AIM-V medium to 4 wells in a 12-well plate, 2 mL per well, and then transfer it to a cell culture incubator at 37°C with 5% CO 2 Preheat for 1 hour;

[0188] 2) Prepare the electroporation solution for single use per well according to the following table:

[0189] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of the solution]]> 82 Electroporation supplement solution 18

[0190] 3) Take the TIL cells obtained in Example 1 into 4 EP tubes, add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL of normal saline, and repeat the centrifugation step to wash the cell pellet;

[0191] 4) Add 5 μg of plasmid pNB328-EGFP to the electroporation solution prepared in 2), and then let it stand at room temperature for within 30 min;

[0192] 5) Resuspend the 4 tubes of TIL cells with the plasmid-containing electroporation solution prepared in 4), 100 μL per tube. Carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette. Place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation chamber, start the electroporation program, and select T-020 for the electroporation program;

[0193] 6) After electroporation, carefully take out the electroporation cuvette, aspirate the cell suspension and transfer it into an EP tube. Add 200 μL of preheated AIM-V medium to each tube, and then transfer it to the wells containing preheated AIM-V medium in the 12-well plate in 1). Incubate at 37°C with 5% CO 2 Culture;

[0194] 7) Add compound H-151 shown in Formula 5 to 3 wells to a final concentration of 0.5 μM at 0 h, 1 h, and 5 h of culture respectively; do not add H-151 to the remaining one well as a control well, and then continue the culture.

[0195] Repeat the culture operation 3 times, and use a cell counter to detect the cell number and cell viability in each well after 5 days of culture, and take the average value.

[0196] The results are as Figures 9-11 shown. Figures 9-11The number of viable cells, the proportion of viable cells, and the proportion of EGFP-positive cells were separately shown in the treatment group with 0.5 μM H-151 added at 0 h, 1 h, and 5 h after electroporating TIL cells with pNB328-EGFP and the control group without H-151 added, after culturing for 5 days. The results showed that adding H-151 molecules at 0 h, 1 h, and 5 h after electroporating TIL cells with the plasmid expressing EGFP could significantly increase the number of viable cells, the proportion of viable cells, and the proportion of cells with positive EGFP expression after electroporation of TIL cells. This indicates that adding the STING inhibitor H-151 can significantly improve the survival level of TIL cells after electroporation and enhance the transfection efficiency of foreign genes.

[0197] Example 6 Application of the STING small molecule inhibitor H-151 in the preparation of T cells overexpressing EGFP by electroporation

[0198] Electroporate T cells expressing EGFP according to the following steps:

[0199] 1) Pre-add 2 mL of AIM-V medium to 4 wells in a 12-well plate, then transfer it to a cell culture incubator and preheat at 37 °C with 5% CO 2 for 1 hour;

[0200] 2) Prepare the electroporation solution according to the single-dose amount per well as shown in the following table:

[0201]

[0202]

[0203] 3) Take the activated T cells obtained in Example 2 into 4 EP tubes, add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cell pellet with 500 μL of normal saline, and repeat the centrifugation step to wash the cell pellet;

[0204] 4) Add 5 μg of plasmid pNB328-EGFP to the electroporation solution prepared in 2), then let it stand at room temperature for within 30 min;

[0205] 5) Resuspend the 4 tubes of T cells with the plasmid-containing electroporation solution prepared in 4), 100 μL per tube, carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette, place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation chamber, start the electroporation program, and select T-020 for the electroporation program;

[0206] 6) After the electroporation is completed, carefully take out the electroporation cup, aspirate the cell suspension and transfer it to an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube, and then transfer it to the well containing pre-warmed AIM-V medium in the 12-well plate in 1). Incubate at 37 °C and 5% CO 2 cultivate;

[0207] 7) Add the compound H-151 shown in Formula 5 to a final concentration of 1 μM to 3 wells after culturing for 0 h, 1 h, and 5 h respectively; do not add H-151 to the remaining well as a control well, and then continue culturing.

[0208] Repeat the culturing operation 3 times, and use a cell counter to detect the cell number and cell viability in each well after culturing for 5 days, and take the average value.

[0209] The results are as Figures 12-14 shown. Figures 12-14 Respectively show the number of live cells, the proportion of live cells, and the proportion of EGFP-positive cells in the treatment group with H-151 added to 1 μM and the control group without H-151 after culturing for 0 h, 1 h, and 5 h after electroporating activated T cells with pNB328-EGFP. The results show that adding the H-151 molecule at 0 h, 1 h, and 5 h after electroporating T cells with the plasmid expressing EGFP can significantly increase the number of live cells and the proportion of cells positive for EGFP expression after electroporating activated T cells. This indicates that adding the STING inhibitor H-151 can significantly improve the survival level of activated T cells after electroporation and improve the transfection efficiency of foreign genes.

[0210] Application of the cGAS small molecule inhibitor IRAK-IN-4 in the preparation of TIL cells overexpressing EGFP by electroporation

[0211] Electroporate TIL cells expressing EGFP according to the following steps:

[0212] 1) Pre-add AIM-V medium to 4 wells in a 12-well plate, 2 mL per well, and then transfer it to a cell culture incubator at 37 °C and 5% CO 2 Preheat for 1 hour;

[0213] 2) Prepare the electroporation liquid ratio for each well once according to the following table:

[0214] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of the solution]]> 82 Electroporation supplement solution 18

[0215] 3) Take the TIL cells obtained in Example 1 into 4 EP tubes, add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, and then resuspend the cell pellet with 500 μL of normal saline, and repeat the centrifugation step to wash the cell pellet;

[0216] 4) Add 5 μg of plasmid pNB328-EGFP to the electrotransformation solution prepared in 2), and then let it stand at room temperature for within 30 min;

[0217] 5) Resuspend 4 tubes of TIL cells with the electrotransformation solution containing the plasmid prepared in 4), 100 μL per tube. Carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette. Place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation chamber, and start the electroporation program. Select program T-020 for the electroporation;

[0218] 6) After the electroporation is completed, carefully take out the electroporation cuvette, aspirate the cell suspension and transfer it into an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube, and then transfer it into the wells containing pre-warmed AIM-V medium in the 12-well plate prepared in 1). Incubate at 37 °C and 5% CO 2 culture;

[0219] 7) Add the cGAS inhibitor IRAK-IN-4 to a final concentration of 2 μM to 3 wells at 0 h, 1 h, and 5 h of culture respectively; do not add IRAK-IN-4 to the remaining one well as a control well, and then continue the culture.

[0220] Repeat the culture operation 3 times, and use a cell counter to detect the cell number and cell viability in each well after 5 days of culture, and take the average value.

[0221] The results are as Figures 15-17 shown. Figures 15-17 Respectively show the number of live cells, the proportion of live cells, and the proportion of EGFP-positive cells in the treatment group with IRAK-IN-4 added to 2 μM at 0 h, 1 h, and 5 h after electrotransformation of TIL cells with pNB328-EGFP and the control group without adding IRAK-IN-4 after 5 days of culture. The results show that adding the IRAK-IN-4 molecule at 0 h, 1 h, and 5 h after electrotransformation of TIL cells expressing EGFP plasmid did not significantly increase the number of live cells, the proportion of live cells, and the proportion of cells with positive EGFP expression after electrotransformation of TIL cells. This indicates that adding the cGAS inhibitor IRAK-IN-4 has no obvious effect on improving the survival level of TIL cells after electrotransformation and enhancing the transfection efficiency of foreign genes.

[0222] Comparative Example 2 Application of the STING small molecule inhibitor C-170 in electrotransformation to prepare T cells overexpressing EGFP

[0223] Electrotransform T cells expressing EGFP according to the following steps:

[0224] 1) Pre-add AIM-V medium to 4 wells in a 12-well plate, 2 mL per well, and then transfer it into a cell culture incubator at 37 °C and 5% CO 2 preheat for 1 hour;

[0225] 2) Prepare the electroporation solution for single use per well according to the following table:

[0226] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of the solution]]> 82 Electroporation supplement solution 18

[0227] 3) Transfer the activated T cells obtained in Example 2 into 4 EP tubes. Add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL of normal saline, and repeat the centrifugation step to wash the cell pellet;

[0228] 4) Add 5 μg of plasmid pNB328-EGFP to the electroporation solution prepared in 2), and then let it stand at room temperature for within 30 min;

[0229] 5) Resuspend the 4 tubes of T cells with the plasmid-containing electroporation solution prepared in 4), 100 μL per tube. Carefully aspirate the cell suspension and transfer it into a LONZA 100 μL electroporation cuvette. Place the electroporation cuvette into the LONZA Nucleofector TM 2b electroporation chamber, start the electroporation program, and select T-020 for the electroporation program;

[0230] 6) After electroporation, carefully take out the electroporation cuvette, aspirate the cell suspension and transfer it into an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube, and then transfer it into the wells containing pre-warmed AIM-V medium in the 12-well plate in 1), and culture at 37 °C and 5% CO 2 culture;

[0231] 7) Add STING inhibitor C-170 to a final concentration of 2 μM to 3 wells at 0 h, 1 h, and 5 h after culture respectively; do not add C-170 to the remaining well as a control well, and then continue the culture.

[0232] Repeat the culture operation 3 times, and detect the cell number and cell viability in each well after 5 days of culture using a cell counter, and take the average value.

[0233] The results are as Figures 18-20 shown. Figures 18-20 Respectively show the number of live cells, the proportion of live cells, and the proportion of EGFP-positive cells in the treatment group with C-170 added to 2 μM at 0 h, 1 h, and 5 h after electroporating activated T cells with pNB328-EGFP and the control group without adding C-170 after 5 days of culture. The results show that adding C-170 molecules at 0 h, 1 h, and 5 h after electroporating T cells with the plasmid expressing EGFP did not significantly increase the number of live cells, the proportion of live cells, and the proportion of cells with positive EGFP expression after electroporation of T cells. This indicates that adding STING inhibitor C-170 has no obvious effect on improving the survival level of activated T cells after electroporation and on improving the transfection efficiency of foreign genes.

[0234] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above content of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of the compound G150 shown in Formula 22 in cultured and electroporated cells, wherein, the cells are immune effector cells selected from one or more of T cells, TIL, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages, Formula 22.

2. The use according to claim 1, characterized in that, the immune effector cells are selected from one or more of T cells, TIL, and CAR-T cells.

3. A method for culturing and electroporating cells, characterized in that, the method comprises the step of culturing the electroporated cells in a culture medium containing the compound G150 shown in Formula 22; the cells are immune effector cells selected from one or more of T cells, TIL, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; after electroporation, the electroporated cells are transferred into the culture medium and cultured for 0 - 5 hours, and the final concentration of the compound G150 shown in Formula 22 in the culture medium is in the range of 0.5 - 5 μM, Formula 22.

4. The method according to claim 3, characterized in that, the method comprises, after electroporation, transferring the electroporated cells into the culture medium and culturing for 0 - 5 hours, then adding the compound G150 shown in Formula 22 to the culture medium and continuing the culture; after adding the compound G150 shown in Formula 22, its final concentration in the culture medium is in the range of 0.5 - 1 μM.

5. The method according to claim 3, characterized in that, the method comprises, after electroporation, transferring the electroporated cells into the culture medium and culturing for 0 - 5 hours, then adding the compound G150 shown in Formula 22 to the culture medium and continuing the culture; after adding the compound G150 shown in Formula 22, its final concentration in the culture medium is in the range of 2.5 - 5 μM.

6. The method according to claim 3, characterized in that, the immune effector cells are selected from one or more of T cells, TIL, and CAR-T cells.

7. The method according to claim 3, characterized in that, the culture medium is a cell culture medium.

8. The method according to claim 7, characterized in that, the culture medium is a culture medium for culturing immune effector cells.

9. The method according to claim 7, characterized in that, the culture medium is selected from any one of GIBCO® AIM-V® CTS™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.

10. The method according to claim 9, characterized in that, the culture medium is GIBCO® AIM-V® CTS™ serum-free cell culture medium.

11. A method for electroporating cells to prepare cells overexpressing a foreign gene, characterized in that, the method comprises: 1) Introducing a nucleic acid containing an expressed foreign gene into the cells by electroporation; 2) Culturing the cells electroporated with the foreign gene in step 1) in a culture medium containing the compound G150 shown in Formula 22; Among them, the electrotransfected cells are cultured for 0 - 5 h; then the compound G150 shown in Formula 22 is added to the medium; after adding the compound G150 shown in Formula 22, its final concentration in the medium ranges from 0.5 - 5 μM. The cells are immune effector cells, selected from one or more of T cells, TIL, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Formula 22.

12. According to the method described in claim 11, wherein, the medium is a medium for culturing immune effector cells; and / or, the electrotransfected cells are cultured for 0 - 1 h; and / or, after adding the compound G150 shown in Formula 22, its final concentration in the medium ranges from 0.5 - 1 μM.

13. As the method described in claim 11, wherein, the medium is a medium for culturing immune effector cells; and / or, the electrotransfected cells are cultured for 0 - 1 h; after adding the compound G150 shown in Formula 22, its final concentration in the medium ranges from 2.5 - 5 μM.

14. According to the method described in claim 12, wherein, the medium is selected from any one of GIBCO® AIM-V® CTS™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.

15. A cell culture medium that can improve the survival rate of cells after electrotransfection, wherein, the cell culture medium is added with the compound G150 shown in Formula 22, and the cells are immune effector cells, selected from one or more of T cells, TIL, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Formula 22.

16. According to the cell culture medium described in claim 15, wherein, the concentration of the compound G150 shown in Formula 22 in the cell culture medium ranges from 0.5 - 5 μM; and / or, the cell culture medium is a medium for culturing immune effector cells.

17. According to the cell culture medium described in claim 16, wherein, the concentration of the compound G150 shown in Formula 22 in the cell culture medium ranges from 0.5 - 2.5 μM; and / or, the cell culture medium is selected from any one of GIBCO® AIM-V® CTS™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.

18. As the cell culture medium described in claim 16, wherein, the concentration of the compound G150 shown in Formula 22 in the cell culture medium ranges from 2.5 - 5 μM; and / or, the cell culture medium is selected from any one of GIBCO® AIM-V® CTS™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.

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