CN111117967B

CN111117967B - Method for preparing cell over expressing exogenous gene

Info

Publication number: CN111117967B
Application number: CN201811287700.8A
Authority: CN
Inventors: 金华君; 徐飞; 黄晨; 刘祥箴; 钱其军
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2023-02-17
Anticipated expiration: 2038-10-31
Also published as: CN111117967A

Abstract

The present invention relates to a method for producing a cell overexpressing a foreign gene. The invention utilizes the STING signal path inhibitor, especially carbonyl cyano 3-chlorophenylhydrazone, to culture and culture the electrotransfer cell, especially the electrotransfer immune effector cell, and can obviously improve the survival rate and the total number of the cells after the cell electrotransfer.

Description

Method for preparing cell over expressing exogenous gene

Technical Field

The present invention relates to the culture of electrically transferred cells, and is especially the process of preparing cell over expressing exogenous gene.

Background

Adoptive Cell Therapy (ACT) using immune effector cells expressing Chimeric Antigen Receptors (CARs), such as CAR-T cells, for treating tumors has the potential to permanently alter the current state of tumor Therapy. Such therapies rely on highly efficient, stable and safe gene transfer platforms. The transfer of synthetic genes encoding chimeric antigen receptors into immune effector cells, such as T cells, is the first step in achieving tumor therapy. Gene transfer techniques include mainly viral and non-viral methods. The viral approach essentially involves the use of retroviral or lentiviral vectors to express the CAR gene, which is introduced into the immune effector cell by packaged viral particles and integrated into the cell genome by the retroviral or lentiviral self-integration system. The advantage of viral vector systems is that the viral particles are able to efficiently transduce immune effector cells, such as T cells, and to efficiently and stably integrate into the host cell genome. However, the preparation and production process of the virus is high in cost, time-consuming and labor-consuming, and in order to meet clinical safety standards, immune effector cells modified by a virus system need to show no virus replication, low genotoxicity and low immunogenicity, and long-term monitoring is needed after the virus is returned to a human body, so that certain potential safety hazards exist.

Electrotransformation (or electroporation) is already a well established method in some areas of medicine, but its use in biotechnology has only recently emerged. By applying a certain high electric field pulse to the cell or tissue instantaneously, permeability is formed on the surface of the cell membrane instantaneously, and charged molecules enter the cell. Classical electroporation will lead to enhanced transport of cells across membranes and altered conductivity. The effect of this process on the cell membrane is related to the intensity, repetition, duration and number of electrical pulses of the electrical transduction. Artificial bilayers, cells or tissues, by their very nature, are already permeabilized by several common electroporation procedures. In electroporation-based transgenic manipulations, foreign DNA is introduced intracellularly by reversible electroporation, and the foreign gene is expressed in its new host cell and inherited as the cell divides. The use of electrotransfer in combination with a non-viral gene modification system capable of inducing stable expression of the transgene, such as a transposon system, is another effective method for modifying immune effector cells in addition to viral vector systems. The transposable subsystem, such as Sleeping Beuty or PiggyBac transposable system, comprises a transposase coding sequence, wherein the coded transposase recognizes repetitive sequences on two sides and can efficiently mediate and integrate into a host cell genome. Transposon systems have enjoyed widespread therapeutic application in combination with electrotransfer and have achieved clinical-level requirements (Kebrieii P, huls H, jena B, munsell M, jackson R, et al (2012) Infusing CD19-Directed T Cells to automatic Disease Control in Patients Undergoid automated Stem-Cell transformation for Advanced B-Cell amplification Maliganic. Human Gene Therapy 23. The use of transposon systems for ACT relies on The electrotransformation of T Cells with Tumor Infiltrating Lymphocytes (TIL), for which there are currently more established commercial electrotransformation instruments and buffers (e.g., lonza Nuclear organisms), and recently there are also reports of methods for The successful construction of CAR-T Cells by commercial electrotransformation systems using SB transposon systems (Jin Z, maiti S, huls H, singh H, olivares S, et al (2011) The genetic modification of T Cell to expression vector, gene Therapy 18-P849-Peng PD, cohen Yam S, cell C, genetic Therapy, PCR 19, protein Gene expression vector 444, and The genetic transformation method for protein expression vector, protein expression, PCR 18-P, PCR 19, protein Gene expression, protein expression vector, PCR 19, protein Gene expression, PCR expression vector, PCR 19, and protein expression vector for DNA expression.

Carbonylcyano 3-chlorophenylhydrazone (CCCP) is a proton carrier (H) ⁺ ionophore) and potent mitochondrial oxidative phosphorylation uncoupler to promote intramitochondrial membrane pairing with H ⁺ Permeability is generated, membrane potential on both sides of the mitochondrial inner membrane is lost, and apoptosis is induced. Can also act on chloroplast membranes to inhibit photosynthesis; CCCP also inhibits protein transport between the endoplasmic reticulum-Golgi apparatus (ER-Golgi), and binds cytochrome C oxidase with high affinity. CCCP also has other functions: 1) Experiments with bullfrog sympathetic nerves have shown that CCCP enhances luteinizing hormone releasing hormone release; 2) Coli cell activity in the presence of glucose as a proton conductor; 3) Various regulatory effects on intracellular calcium levels; 4) Inhibiting the secretion of fatty liver enzymes; 5) Cl partially inhibiting pH gradient activation ^- Uptake and brushing of marginal Membrane Cl ^- /Cl ^- Swapping, etc. Recent reports have also shown that CCCP is capable of causing dissipation of mitochondrial membrane potential and further inhibiting the STING pathway of cells (D.Prantner, D.J.Perkins, W.Lai, M.S.Williams, S.Sharma, K.A.Fitzgerald, S.N.Vogel,5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activators stimulant of interferon gene (STING) -dependent agonist pathway and isgulated by mitochondrial membrane potential,J.Biol.Chem.287(2012))。

Compared with a viral vector system, the electrotransfer combined transposable system has the advantages of simple operation, low immunogenicity and genotoxicity and low safety risk, and is an important method for ACT. But the problems existing in the method are also obvious: excessive cell death is easily caused at a transient high voltage, and transfection efficiency is low, particularly in relation to the cell type and the electrotransfer conditions (including voltage, waveform, pulse time and electrotransfer buffer composition). Especially for immune effector cells such as PBMC and TIL cells, the difficulty of electrotransformation is relatively larger, and although mature electrotransformation instruments and matched buffer systems are already available on the market at present, the problem of high mortality rate of the immune effector cells after electrotransformation is still more prominent. There is therefore still a need for a method of increasing the survival rate of immune effector cells after electrotransformation.

Disclosure of Invention

In a first aspect, the invention provides the use of an inhibitor of the STING signalling pathway in culturing an electrotransfer cell.

In a second aspect, the present invention provides a method of culturing an electrotransferred cell, the method comprising the step of culturing the electrotransferred cell in a medium comprising an inhibitor of the STING signaling pathway.

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

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

In one or more embodiments, the nucleic acid capable of inhibiting the STING signaling pathway comprises an siRNA, antisense RNA, ribozyme, gene editing vector such as CRISPR-CAS9 gene editing vector or TALEN gene editing vector that targets the STING signaling pathway or reduces or eliminates mitochondrial membrane potential.

In one or more embodiments, the small molecule compound capable of inhibiting STING signaling pathway includes small molecule compounds capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, including but not limited to carbonylcyano 3-chlorophenylhydrazone (CCCP).

In one or more embodiments, the use is of carbonylcyano 3-chlorophenylhydrazone in culturing an electroporated immune effector cell.

In one or more embodiments, the method is a method of culturing an electroporated immune effector cell, the method comprising the step of culturing the electroporated immune effector cell in a medium comprising a carbonylcyano 3-chlorophenylhydrazone.

In one or more embodiments, the method or use comprises transferring the electroporated cells into a culture medium for 0.5-8 hours after the end of electroporation, transferring the STING signaling pathway inhibitor into the culture medium, and continuing the culturing.

In one or more embodiments, the cell is an immune effector cell.

In one or more embodiments, the immune effector cell is selected from one or more of a T cell, a TIL cell, an NK T cell, a CAR-T cell, a CIK cell, a TCR-T cell, and a macrophage.

In one or more embodiments, the immune effector cell is selected from one or more of a T cell, a TIL cell, and a CAR-T cell.

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

In one or more embodiments, the medium is a medium of T cells, TIL cells, or CAR-T cells.

In one or more embodiments, the medium is selected from

CTS ^TM Any one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, is

CTS ^TM Serum-free cell culture media.

In one or more embodiments, the final concentration of the STING signaling pathway inhibitor in the medium is in the range of 5-100 μ M, preferably 10-80 μ M.

The invention also provides a cell culture medium containing 5-100. Mu.M, preferably 10-80. Mu.M, of a STING signalling pathway inhibitor.

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

In one or more embodiments, the medium used to culture immune effector cells is a medium of T cells, TIL cells, or CAR-T cells.

In one or more embodiments, the medium used to culture the immune effector cells is selected from

CTS ^TM Serum-free cell culture media.

Drawings

FIG. 1: after electroporation, CCCP was added to the TIL-single-transfer cell wells for 1h and 5h, and the total number of cells in each group was determined after 5 days of incubation.

FIG. 2: after electroporation, CCCP was added to the TIL-single-transfer cell wells for 1h and 5h, and the total number of viable cells in each group was determined after 5 days of incubation.

FIG. 3: after electroporation, CCCP was added to TIL-double-rotating cell wells for 1h and 5h, and the number of cells in each group was counted after 3 days of culture.

FIG. 4: CCCP was added to TIL-double-rotating

cell wells

1h and 5h after electroporation, and the number of EGFP-positive cells was cultured 7 days later.

FIG. 5: after 1h and 5h of culturing after electroporation CCCP was added to the TIL-double rotating cell wells, the total number of cells after 14 days of culturing.

FIG. 6: after the electrotransfer, CCCP is added into TIL-double rotating cell holes after 1h and 5h of culture, and the proportion of living cells after 14 days of culture is obtained.

FIG. 7: after the electroporation, CCCP was added to the TIL-double-rotating cell wells for 1 hour and 5 hours, and the number of viable cells was measured after 14 days of culturing.

FIG. 8: after electrotransfer culture for 0.5h, 1h and 5h CCCP was added to the T-cell single transfer wells and the total number of cells cultured for 14 days.

FIG. 9: after electrotransfer, 0.5h, 1h and 5h of culture were added to the CCCP to the T-cell single transfer wells, and the proportion of viable cells was found after 14 days of culture.

FIG. 10: shows that after 1h of culture after electrotransfer, CCCP is added into T-single transfer cell wells, and the total number of cells after 14 days of culture is obviously more than that of a control group; the CCCP is added 0.5h before the electric transfer and then the electric transfer is carried out, the total number of the cells obtained after the 14 days of culture is higher than that of the control group, but still is obviously lower than that obtained after the CCCP is added 1h after the electric transfer.

FIG. 11: adding CCCP 0.5h before electric transfer, electric transfer to T-single transfer cell hole, and adding CCCP 1h after electric transfer, and culturing for 14 days.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described below (e.g., examples) may be combined with each other to constitute a preferred embodiment.

Interferon activating protein (STING) is an important molecule in innate immune response and plays an important role in defense against viral and intracellular bacterial infections and in mediating the production of type I interferons. STING, an important component of the signal transduction cascade, can exert immune defense effects in the case of infection with pathogens (viruses, bacteria, parasites, etc.) as well as anti-tumor immune responses in the case of tumor development. When the STING signaling pathway is over-activated, a range of autoimmune diseases may also be caused.

The present inventors have found that the survival rate of cells after electrotransfer can be significantly improved by culturing cells, particularly immune effector cells, after the electrotransfer of the cells, particularly immune effector cells, is terminated and the cells are transferred to a culture medium for a period of time and then adding a STING signal pathway inhibitor for culturing.

Herein, the cell may be any cell of interest, particularly cells conventionally used in the art for electroporation to express foreign genes, and may be eukaryotic cells (e.g., animal cells and plant cells) and prokaryotic cells (e.g., E.coli and other bacteria, etc.). For example, the cell can be a human cell. Examples of cells include, but are not limited to, HEK293 cells, MDCK cells, hela cells, and the like. In certain embodiments, the cell is an immune cell. Herein, immune effector cells refer to immune cells involved in the clearance of foreign antigens and the functioning of effectors in an immune response, including but not limited to one or more of T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Preferably, in certain embodiments, the immune effector cells suitable for use in the methods of the invention are selected from one or more of T cells, TILs and CAR-T cells.

Herein, the STING signaling pathway inhibitor can be any of various agents known in the art that inhibit the STING signaling pathway, including but not limited to proteins, nucleic acids, and small molecule compounds that inhibit the STING signaling pathway. For example, proteins capable of inhibiting STING signaling pathways include proteins that inhibit STING expression and/or binding activity or that reduce or eliminate mitochondrial membrane potential, including but not limited to ULK1, ULK2, caspase3, caspase7, and caspase9. Nucleic acids capable of inhibiting the STING signaling pathway include, but are not limited to, sirnas, antisense RNAs, ribozymes, and gene editing vectors, such as CRIPR-CAS9 gene editing vectors or TALEN gene editing vectors, that target the STING signaling pathway or reduce or eliminate mitochondrial membrane potential. Small molecule compounds capable of inhibiting STING signaling pathways include, but are not limited to, small molecule compounds capable of inhibiting STING expression and/or binding activity or small molecule compounds that reduce or eliminate mitochondrial membrane potential, including, but not limited to, carbonylcyano 3-chlorophenylhydrazone (CCCP). In certain embodiments, the invention relates to the use of CCCPs for culturing transfected cells.

Electroporation, also known as electroporation, is used to introduce the DNA of interest into the host cell. The invention can be practiced using various electrotransformation methods and agents known in the art, e.g., LONZA can be used

I Device and electrotransformation reagent provided by it. The electrotransformation liquid can be prepared according to the instruction of a commercially available electrotransformation reagent, and electrotransformation plasmid is added. When the cells are electroporated, the cells are resuspended in an electroporation solution containing an electroporation plasmid, and electroporation is carried out in an electroporation apparatus.

The electrotransfer plasmid may be any plasmid of interest, in particular a plasmid expressing a Chimeric Antigen Receptor (CAR). The amount of electrotransport plasmid may be an amount conventional in the art, for example, every 5X 10 ⁶ Cells were electroporated with 3-8ug of plasmid.

After the end of the electrotransfer, the cell suspension is removed, preheated cell culture medium is added and the CO is then reduced under conventional conditions (e.g.37 ℃ C., 5% ₂ ) And (4) culturing. After at least 0.5 hour of incubation, STING signaling pathway inhibitors (particularly carbonylcyano 3-chlorophenylhydrazone) were added to the cell culture medium containing the transfected cells. Addition of STING signaling pathway inhibitors either prematurely or too late may not increase the total number and survival of the electroporated cells. Therefore, it is preferable to add the STING signaling pathway inhibitor (particularly, carbonylcyano 3-chlorophenylhydrazone) to the medium within 8 hours, more preferably within 5 hours, even more preferably within 3 hours of culturing the transfected cells. For example, in certain particularly preferred embodiments, the STING signaling pathway inhibitor is added at 0.5-3 hours of culturing the transduced cells, preferably at 45min to 2 hours of culturing, and more preferably at 1-2 hours of culturing.

In general, the final concentration of the STING signaling pathway inhibitor (especially carbonylcyano 3-chlorophenylhydrazone) in the post-addition medium is in the range of 5-100. Mu.M, preferably in the range of 10-80. Mu.M.

Herein, the cell culture medium may be various media suitable for the culture of cells, particularly immune cells, particularly media conventionally used in the art for culturing various electroporated cells. In certain embodiments, the medium is a medium for culturing immune cells, including but not limited to for culturing T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cellsAnd a culture medium for one or more of macrophages. In certain embodiments, the culture medium is a culture medium of T cells, TILs and/or CAR-T cells, in particular a culture medium that cultures transduced T cells, TILs and/or CAR-T cells. Exemplary culture media include, but are not limited to

CTS ^TM Any one of serum-free cell culture medium, DMEM medium and RPMI1640 medium; preferably, is

CTS ^TM Serum-free cell culture media.

After the electroporation is cultured according to the conventional culture process, the total cell number and/or the survival rate of the electroporation can be obviously improved by the culture with the addition of the STING signaling pathway inhibitor compared with the conventional electroporation in the field.

Accordingly, the present invention provides a method for culturing an electrotransferred cell (particularly, an immune effector cell), which comprises transferring the electrotransferred cell to a culture medium after the completion of the electrotransferred cell for 0.5 to 8 hours, adding a STING signaling pathway inhibitor (particularly, carbonylcyano 3-chlorophenylhydrazone) to the culture medium, and continuing the culture. Preferably, the culture is carried out by adding the STING signal pathway inhibitor (especially carbonylcyano 3-chlorophenylhydrazone) when the culture medium is cultured for 0.5-5h, 0.5-3h, 0.5-2h or 1-2 h.

In certain embodiments, the present invention provides a method of electrotransformation of cells expressing a foreign gene (particularly immune effector cells), the method comprising:

1) Introducing a nucleic acid containing an expressed foreign gene into a cell by electrotransfer;

2) Culturing 1) the cells transfected with the foreign gene in a medium containing a STING signaling pathway inhibitor (particularly, carbonylcyano 3-chlorophenylhydrazone);

wherein the STING signaling pathway inhibitor is added after the completion of the electrotransformation when the electrotransferred cells are cultured for 0.5-8h, preferably 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2 h.

The invention also provides the use of STING signalling pathway inhibitors (especially carbonylcyano 3-chlorophenylhydrazone) in the culture of transduced cells (especially immune effector cells).

The invention also provides a cell culture medium containing the STING signaling pathway inhibitor. Preferably, the cell culture medium is a medium for immune effector cells. More preferably, the STING signaling pathway inhibitor is CCCP. Thus, in certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells supplemented with carbonylcyano 3-chlorophenylhydrazone, such as a medium conventionally used for culturing one or more of T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages, particularly T cells, TIL cells, and/or CAR-T cells. More preferably, the medium is a medium conventionally used for culturing electroporated immune effector cells. The carbonyl cyano-3-chlorophenylhydrazone is added into the culture medium and used for culturing the immune effector cells after the electrotransformation, so that the total number and the survival rate of the cells can be obviously improved. Preferably, the final concentration of carbonylcyano 3-chlorophenylhydrazone in the medium is in the range of 5-100. Mu.M, preferably in the range of 10-80. Mu.M. An exemplary immune effector cell culture medium of the present invention is carbonyl cyano 3-chlorophenylhydrazone-containing

CTS ^TM Serum-free cell culture medium, DMEM medium or RPMI1640 medium; preferably, it is a compound containing carbonylcyano 3-chlorophenylhydrazone

CTS ^TM Serum-free cell culture media; more preferably carbonyl cyano 3-chlorophenylhydrazone in the above concentration

CTS ^TM Serum-free cell culture media.

The invention has the advantages that the death rate of the cells after the electrotransformation can be obviously reduced and the number of the live cells and the proportion of the live cells after the cells are electrically transformed can be improved by culturing the cells after the electrotransformation, especially immune effector cells, by using the culture medium containing the STING signal pathway inhibitor, such as carbonyl cyano 3-chlorophenylhydrazone (CCCP).

The invention will be elucidated hereinafter by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The electrotransformation apparatus used in the examples was a LONZA Nucleofector from Lonza ^TM 2b; PBMCs used in the examples were purchased from ALLCELLS corporation, cat #: PB003F-C. Other methods and reagents used in the examples are conventional in the art.

Example 1: isolated culture of liver cancer tissue-derived TIL cells

Freshly excised liver cancer specimens were collected and immediately processed under sterile conditions. The specific method comprises the following steps: removing normal tissue and necrotic area around the liver cancer specimen, and removing 1-2mm size from different areas of the specimen ³ One for each well of a 24-well plate. 2mL of complete medium (GT-T551 medium containing 10% FBS) and 3000IU/mL of IL-2 were added per well. Placing 24-well plate at 37 deg.C, 5% ₂ Culturing in an incubator. Half-volume changes were made for all wells on days 5-6 after initiation of culture. And then, according to the growth condition of the TIL, half-amount liquid change is carried out every 1-2 days. Once the wells are full of TIL and all adherent cells have been removed, the TIL from each full well is collected.

Example 2: construction of recombinant expression vectors

The EGFP coding sequence was artificially synthesized and inserted between EcoRI and SalI cleavage sites of PB transposable system-based vectors pNB328 and pS328 (the structure and sequence of pNB328 is described in CN201510638974.7, which is incorporated herein by reference in its entirety; pS328 lacks the PB transposon sequence as compared to pNB 328; the EGFP coding sequence is shown in SEQ ID NO:9 in CN 201510638974.7), and the constructed recombinant expression vectors were named pNB328-EGFP and pS328-EGFP, respectively.

The coding sequence of PB transposase was artificially synthesized and inserted between EcoRI and SalI cleavage sites of pS328 based on the PB transposase system, the coding sequence of PB transposase is shown in SEQ ID NO:5 in CN201510638974.7, and the constructed recombinant expression vector was named pS328-PB.

Example 3: preparation of EGFP-expressing TIL (TIL-Single transfer plasmid) by electric transfer of pNB328-EGFP

Electrically converting the TIL of the EGFP expression into a single conversion according to the following steps:

1) AIM-V medium was previously added to 3 wells of 12-well plates, nos. a, b, c, 2mL per well, and then transferred into a cell incubator at 37 ℃ 5% ₂ Preheating for 1 hour;

2) The electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL Nucleocuvette ^TM Strip(μL)
		Nucleofector ^TM volume of solution	82
Electrotransformation make-up solution	18

3) Taking the TIL obtained in example 1 into 3 EP tubes, 5X 10 tubes were added into each EP tube ⁶ Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. Mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) Adding plasmid pNB328-EGFP 6 mug into the electrotransformation liquid prepared in the step 2), and then standing for 30min at room temperature;

5) By 4) Suspending 3 tubes of TIL (100 μ L per tube) of the prepared plasmid-containing electrotransfer solution carefully, sucking the cell resuspension solution into a LONZA 100 μ L electric rotating cup, and placing the electric rotating cup into a LONZA Nucleofector ^TM 2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects X001;

6) Carefully removing the electric rotary cup after completing the electric transfer, pipetting the cell suspension and transferring to EP tubes, adding 200. Mu.L of preheated AIM-V medium to each tube, and transferring to three wells a, b, and c containing preheated AIM-V medium in 12-well plate of 1), 37 ℃,5% CO ₂ Culturing;

7) Adding CCCP to the a-well and the b-well at the time points of culturing for 1h and 5h respectively to a final concentration of 10 μ M; wells were not added CCCP, control wells, and then culture was continued.

The culturing operation was repeated 3 times, and the number of cells in each well after 5 days of culturing was counted and averaged, and the results are shown in FIG. 1. The results in FIG. 1 show that the total number of cells after 5 days of culture was significantly greater than the control group when CCCP was added to the TIL-single-rotor cell wells at 1h and 5h post-electroporation.

A small amount of trypan blue was stained from the cells in each well after 5 days of culture, and the number of viable cells was counted, as shown in FIG. 2.

The results in FIG. 2 show that the total number of viable cells after 5 days of culture was significantly greater than the control group when CCCP was added to the TIL-single-rotor wells at 1h and 5h post-electroporation.

Example 4: transformation of pS328-EGFP and pS328-PB TIL expressing EGFP was prepared (TIL-double-plasmid)

The procedure for the preparation of EGFP-expressing TIL-double-transgenic cells was as described in example 3, steps 1) -8), except that 1X 10 cells were added to each EP tube in step 2) ⁷ A cell; the same as in example 3 except that in step 4), plasmids pS328-EGFP and pS328-PB were added in an amount of 4. Mu.g each, and the final concentration of CCCP in step 8) was 50. Mu.M.

The number of TIL-duplexes in each well was observed after 3 days of culture and the results are shown in FIG. 3. The results in FIG. 3 show that the number of cells after 3 days of culture was significantly greater than the control group when CCCP was added to TIL-double-rotating cell wells at 1h and 5h post-electroporation.

The luminescence of EGFP was observed in each well after 7 days of culture, and the results are shown in FIG. 4. FIG. 4 shows that after 1h of incubation after electroporation CCCP was added to the TIL-double-rotating cell wells, the number of EGFP-positive cells after 7 days of incubation was comparable to the control group, higher than the TIL-double-rotating-5 h group.

The culturing operation was repeated 3 times, and the number of cells in each well after 14 days of culturing was counted and averaged, and the results are shown in FIG. 5. FIG. 5 shows that the total number of cells after 14 days of culture was significantly greater than the control group by adding CCCP to TIL-double-rotating cell wells 1h after electroporation; after 5h of culture after electroporation, CCCP was added and the total number of cells was about the same as that of the control group after 14 days of culture.

Viable cells were counted after staining with trypan blue, a small amount of suspension, from the cells in each well after 14 days of culture, and the results are shown in FIGS. 6 and 7. FIG. 6 shows that the addition of CCCP to TIL-double-rotating cell wells at 1h and 5h after electroporation resulted in a significantly greater proportion of viable cells after 14 days of culture than the control; FIG. 7 shows that the number of viable cells after 14 days of culture was significantly greater than the control group when CCCP was added to TIL-double-rotor wells at 1h and 5h after electroporation.

In FIGS. 5-7, "con" represents a control and indicates wells not added with CCCP after the electrotransfer, "1h" represents wells added with CCCP 1h after the electrotransfer, and "5h" represents wells added with CCCP 5h after the electrotransfer.

Example 5: generation of EGFP-expressing T cells by electrotransfer of pNB328-EGFP (T cells-Single transfer plasmid)

The procedure for preparing EGFP-expressing T-cell single-transfer cells was the same as in steps 1) to 7) of example 3 except that the cells were changed from TIL to PBMC, four wells a, b, c, and d were set in step 6), and the CCCP was added at the time point of step 7) after 0.5 hour of the electroporation culture, and the rest was the same as in example 3.

The culturing operation was repeated 3 times, and the number of cells in each well after 14 days of culturing was counted and averaged, and the results are shown in FIG. 8. FIG. 8 shows that CCCP was added to T-single-transfer cell wells 1h after electroporation, and that the total number of cells was significantly greater after 14 days of culture than in the control group; the total number of cells cultured for 14 days after the CCCP is added after the culture for 5 hours after the electrotransfer is performed is approximately the same as that of the control group; the total number of cells obtained by culturing for 14 days after adding CCCP after culturing for 0.5h after electrotransfer is more than the number of cells obtained by adding CCCP for 5h, but is still obviously less than the number of cells obtained by adding CCCP for 1 h.

Viable cells were counted after staining with trypan blue, a small amount of suspension, from the cells in each well after 14 days of culture, and the results are shown in FIG. 9. FIG. 9 shows that the proportion of viable cells after 0.5h of incubation after electroporation and 14 days of CCCP addition was substantially the same as the control group, the proportion of viable cells after 1h of incubation after electroporation and 14 days of CCCP addition was significantly higher than the control group, and the proportion of viable cells after 5h of incubation after electroporation and 14 days of CCCP addition was significantly lower than the control group.

In FIGS. 8 and 9, "con" represents a control and indicates a well to which CCCP was not added after the electric transfer, "1h" represents a well to which CCCP was added 1h after the electric transfer, "5h" represents a well to which CCCP was added 5h after the electric transfer, and "0.5h" represents a well to which CCCP was added 0.5h after the electric transfer.

Comparative example 1: preparation of EGFP-expressing T cells by addition of CCCP before and after electroporation (T cells-Single transfection plasmid)

The procedure was the same as in example 3, steps 1) to 7), in which TIL was changed to PBMC cells, and the time point for addition of CCCP in step 7) was set to 0.5h before electroporation and 1h after electroporation, and the rest was the same as in example 3.

The culturing operation was repeated 3 times, and the number of cells in each well after 14 days of culturing was counted and averaged, and the results are shown in FIG. 10. FIG. 10 shows that the total number of cells after 14 days of culture was significantly greater than that of the control group by adding CCCP to T-single-transfer cell wells at 1h after electroporation; the CCCP is added 0.5h before the electric transfer and then the electric transfer is carried out, the total number of the cells obtained after the 14 days of culture is higher than that of the control group, but still is obviously lower than that obtained after the CCCP is added 1h after the electric transfer.

Viable cells were counted after staining with trypan blue, a small amount of suspension, from the cells in each well after 14 days of culture, and the results are shown in FIG. 11. FIG. 11 shows that the ratio of viable cells after 14 days of culture was substantially the same as that of the control group when CCCP was added 0.5h before the electroporation, and that the ratio of viable cells after 14 days of culture after 1h of incubation after the electroporation was significantly higher than that of the control group when CCCP was added.

In FIGS. 10 and 11, "con" represents a control and indicates wells not added with CCCP after electroporation, "1h after electroporation" indicates wells added with CCCP 1h after electroporation, "0.5h before electroporation" indicates wells added with CCCP 0.5h before electroporation.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the above disclosure of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

Use of a sting signalling pathway inhibitor in culturing electrotransfer cells.
2. The use of claim 1, wherein the STING signaling pathway inhibitor is carbonylcyano 3-chlorophenylhydrazone.
3. The use of claim 1 or 2, wherein the cell is an immune effector cell.
4. A method of culturing an electrotransferred cell, comprising the step of culturing the electrotransferred cell in a medium comprising an inhibitor of the STING signaling pathway.
5. The method of claim 4, wherein the STING signaling pathway inhibitor is carbonylcyano 3-chlorophenylhydrazone.
6. The method of claim 4, wherein the cell is an immune effector cell.
7. The method according to any one of claims 4 to 6, wherein the method comprises, after the completion of the electrotransformation, transferring the electroporated cells into a culture medium for 0.5 to 8 hours, and then adding the STING signaling pathway inhibitor to the culture medium and continuing the culture.
8. The method of claim 7, wherein the final concentration of the STING signaling pathway inhibitor in the culture medium after addition is in the range of 5-100 μ Μ.
9. The method of claim 8, wherein the final concentration of the STING signaling pathway inhibitor in the culture medium after addition is in the range of 10-80 μ Μ.
10. The method of any one of claims 4-6, wherein the immune effector cell is selected from one or more of a T cell, a TIL, an NK cell, an NK T cell, a CAR-T cell, a CIK cell, a TCR-T cell, and a macrophage.
11. The method of claim 10, wherein the immune effector cell is selected from one or more of a T cell, a TIL, and a CAR-T cell.
12. The method of any one of claims 4 to 6, wherein the culture medium is a cell culture medium.
13. The method of claim 12, wherein the medium is a medium for culturing immune effector cells.
14. The method of claim 12, wherein the culture medium is selected from the group consisting of

CTS ^TM Serum-free cell culture medium, DMEM medium and RPMI1640 medium.
15. The method of claim 12, wherein the culture medium is

CTS ^TM Serum-free cell culture media.
16. A method for preparing a cell expressing a foreign gene by electroporation, the method comprising:

1) Introducing a nucleic acid containing an expressed foreign gene into a cell by electrotransfer;

2) Culturing the cells transfected with the foreign gene in step 1) with a medium containing a STING signaling pathway inhibitor;

wherein the transfected cells are cultured for 0.5-8h, and then the STING signaling pathway inhibitor is added to the medium.
17. The method of claim 16, wherein the STING signaling pathway inhibitor is carbonylcyano 3-chlorophenylhydrazone.
18. The method of claim 16, wherein the cell is an immune effector cell.
19. The method of claim 16, wherein the STING signaling pathway inhibitor is added to the culture medium after culturing the transduced cells for 0.5-5 hours.
20. The method of claim 16, wherein the STING signaling pathway inhibitor is added to the culture medium after culturing the electroporated cells for 0.5-3 hours.
21. The method of claim 16, wherein the STING signaling pathway inhibitor is added to the culture medium after culturing the electroporated cells for 0.5-2 hours.
22. The method of claim 16, wherein the STING signaling pathway inhibitor is added to the culture medium 1-2 hours after culturing the electroporated cells.
23. The method of claim 16, wherein the final concentration of the STING signaling pathway inhibitor in the culture medium after addition is in the range of 5-100 μ Μ.
24. The method of claim 23, wherein the final concentration of the STING signaling pathway inhibitor in the culture medium after addition is in the range of 10-80 μ Μ.
25. The method of claim 16, wherein the cell is an immune effector cell selected from one or more of the following: t cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.
26. The method of claim 25, wherein the cell is selected from one or more of a T cell, a TIL, and a CAR-T cell.
27. The method of claim 16, wherein the medium is a medium for culturing immune effector cells.
28. The method of claim 27, wherein the culture medium is selected from the group consisting of

CTS ^TM Serum-free cell culture medium, DMEM medium and RPMI1640 medium.
29. The method of claim 27, wherein the method further comprises the step of determining the target position of the target position by using a reference position of the target positionThe culture medium is

CTS ^TM Serum-free cell culture media.
30. The application of the culture medium added with the STING signal path inhibitor in culturing the cells after electric transformation to reduce the death rate of the cells after electric transformation and improve the number of live cells and the proportion of the live cells after electric transformation of the cells.
31. The use according to claim 30, wherein the concentration of the STING signalling pathway inhibitor in the cell culture medium is from 5 to 100 μ M.
32. The use according to claim 31, wherein the concentration of the STING signalling pathway inhibitor in the cell culture medium is from 10 to 80 μ M.
33. The use of claim 30, wherein the STING signaling pathway inhibitor is carbonylcyano 3-chlorophenylhydrazone.
34. The use according to any one of claims 30 to 33, wherein the cell culture medium is a medium for culturing immune effector cells.
35. The use of claim 34, wherein the cell culture medium is selected from the group consisting of

CTS ^TM Serum-free cell culture medium, DMEM medium, and RPMI1640 medium.
36. The use of claim 35, wherein the cell culture medium is

CTS ^TM Serum-free cell culture media.
37. The use of claim 30, wherein the cell is an immune effector cell.
38. The use of claim 30, wherein the cell is an immune effector cell selected from one or more of: t cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.
39. The use of claim 30, wherein the cell is selected from one or more of a T cell, a TIL and a CAR-T cell.