CN111349615B

CN111349615B - Method for preparing cell over-expressing exogenous gene

Info

Publication number: CN111349615B
Application number: CN201811581441.XA
Authority: CN
Inventors: 金华君; 徐飞; 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: 2018-12-24
Filing date: 2018-12-24
Publication date: 2024-08-13
Anticipated expiration: 2038-12-24
Also published as: CN111349615A

Abstract

The present invention relates to a method for preparing a cell over-expressing a foreign gene. The invention utilizes caspase inhibitors to culture electrotransport cells, especially electrotransport immune effector cells, and can obviously improve the survival rate and total number of cells after electrotransport.

Description

Method for preparing cell over-expressing exogenous gene

Technical Field

The invention relates to the culture of electrotransport cells, in particular to a method for preparing cells which overexpress exogenous genes.

Background

Adoptive cell therapy (Adoptive CELL THERAPY, ACT) using Chimeric Antigen Receptor (CAR) expressing immune effector cells, such as CAR-T cells, for treating tumors has the potential to permanently alter the current state of tumor therapy. Such treatments rely on 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 nonviral methods. Viral methods mainly include the use of retroviral or lentiviral vectors to express the CAR gene, introducing the CAR gene into immune effector cells via packaged viral particles, and integrating into the cell genome via the integration system of the retrovirus or lentivirus itself. The advantage of viral vector systems is that the viral particles are able to transduce immune effector cells, such as T cells, efficiently and stably into the host cell genome. However, the preparation and production process of the virus has high cost, time and labor are wasted, and in order to meet clinical safety standards, the immune effector cells transformed by the virus system are required to show no replication, low genotoxicity and low immunogenicity of the virus, and long-term monitoring is required after the virus is returned into a human body, so that certain potential safety hazard exists.

Electrotransformation (or electroporation) is already a well-established method in the field of medicine, but its use in biotechnology has just emerged. By instantaneously applying a certain high electric field pulse to cells or tissues, permeation is instantaneously formed on the surface of cell membranes, thereby leading charged molecules to enter the cells. Classical electroporation will lead to enhanced transport of cells across the membrane and change the 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 transitions. Artificial bilayers, cells or tissues, depending on their own specific properties, can be permeabilized by several commonly used electrotransport procedures. In electroporation-based transgenic procedures, exogenous DNA is introduced into the cell by reversible electroporation, and exogenous genes are expressed in their new host cells and inherited as the cells divide. Electrotransformation in combination with a non-viral gene modification system, such as a transposon system, capable of inducing stable expression of the transgene is another effective method for modifying immune effector cells outside of the viral vector system. Transposon systems, such as Sleeping Beuty or the PiggyBac transposition system, comprise transposase coding sequences that encode transposases that recognize flanking repeats and mediate integration into the host cell genome with high efficiency. The transposon subsystem has a wide therapeutic application prospect in combination with electrotransformation and has completed the clinical-grade requirement (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), that has higher efficiency in T cells. ACT using a transposon system relies on electrotransformation of T cells and tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocyte, TIL), for which there are currently commercially available electrotransformation instruments and matched buffers (e.g. Lonza Nucleofactor) that are relatively mature in the market, recently there is also report (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 a chimeric 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 Therapy16:1042–1049), on a method of successfully constructing CAR-T cells by a commercial electrotransformation system using a SB transposon system and the ACT method using a 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).

Compared with a viral vector system, the electric transfer binding transposon system has the advantages of simplicity in operation, low immunogenicity and genotoxicity and low safety risk, and is an important method for ACT. But the problems are also obvious: excessive cell death is readily caused at transient high voltages and transfection efficiency is low, particularly with respect to cell type and electrotransport conditions (including voltage, waveform, pulse time and electrotransport buffer composition). Particularly, for immune effector cells such as PBMC and TIL cells, the difficulty of electrotransformation is relatively higher, and although a mature electrotransformation instrument and a matched buffer system are already available in the market at present, the problem of high death rate of the immune effector cells after electrotransformation is still more remarkable. There is thus still a need for a method that can increase the survival rate of immune effector cells after electrotransformation.

Disclosure of Invention

The invention provides an application of a caspase inhibitor in culturing electrotransport cells.

In preferred embodiments, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitor, caspase-2 inhibitor, caspase-3 inhibitor, caspase-4 inhibitor, caspase-5 inhibitor, caspase-6 inhibitor, caspase-7 inhibitor, caspase-8 inhibitor, and caspase-10 inhibitor.

In a further preferred embodiment, the caspase inhibitor is a caspase-1 inhibitor, a caspase-3 inhibitor, or a caspase-7 inhibitor.

In a further preferred embodiment, the caspase inhibitor is:

And/or

In a preferred embodiment, the cell is an immune effector cell.

The invention also provides a method of culturing electrotransformed cells comprising the step of culturing the electrotransformed cells in a medium comprising a caspase inhibitor.

In a further preferred embodiment, the caspase inhibitor is:

And/or

In a preferred embodiment, the cell is an immune effector cell.

In a preferred embodiment, the method comprises, after the electrotransfer is completed, transferring the electrotransferred cells into a medium for culturing for 0.5 to 5 hours, and then adding the caspase inhibitor to the medium for further culturing.

In a further preferred embodiment, the final concentration of the caspase inhibitor in the medium after addition is in the range of 5-100. Mu.M, preferably 10-80. Mu.M.

In preferred embodiments, the immune effector cell is selected from one or more of T cells, TIL, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.

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

In a preferred embodiment, the medium is a cell culture medium, preferably a medium for culturing immune effector cells; preferably selected fromCTS ^TM serum-free cell culture medium, DMEM medium, and RPMI1640 medium; more preferablyCTS ^TM serum-free cell culture medium.

The present invention also provides a method for preparing a cell expressing a foreign gene by electrotransformation, the method comprising:

1) Introducing a nucleic acid comprising an expressed exogenous gene into the cell by electrosteering;

2) Culturing the cells having the exogenous gene electrotransferred in step 1) with a medium containing a caspase preparation;

Wherein the caspase inhibitor is added to the culture medium after culturing the electrotransformed cells for 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2h, and culturing is continued.

In a preferred embodiment, the final concentration of the caspase inhibitor in the medium after addition is in the range of 5-100. Mu.M, preferably 10-80. Mu.M.

In a further preferred embodiment, the caspase inhibitor is:

And/or

In a preferred embodiment, the cell is an immune effector cell.

In a preferred embodiment, the medium is a medium for culturing immune effector cells; preferably selected fromCTS ^TM serum-free cell culture medium, DMEM medium, and RPMI1640 medium; more preferablyCTS ^TM serum-free cell culture medium.

The invention also provides a cell culture medium, which is added with the caspase inhibitor.

In a preferred embodiment, the concentration of caspase inhibitor in the cell culture medium is between 5 and 100. Mu.M, preferably between 10 and 80. Mu.M.

In preferred embodiments, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitor, caspase-2 inhibitor, caspase-3 inhibitor, caspase-4 inhibitor, caspase-5 inhibitor, caspase-6 inhibitor, caspase-7 inhibitor, caspase-8 inhibitor, and caspase-10 inhibitor

In a further preferred embodiment, the caspase inhibitor is:

And/or

In a preferred embodiment, the cell culture medium is a medium for culturing immune effector cells, preferably selected fromCTS ^TM serum-free cell culture medium, DMEM culture medium, and RPMI1640 culture medium; more preferablyCTS ^TM serum-free cell culture medium.

Drawings

FIGS. 1A-1C: cell images taken on day 6 of culture of samples of pS328-PD-1scFv and pS328-PB were double-transferred to TIL cells. FIG. 1A is a cell image of a control group (normal electrotransfer culture), FIG. 1B is a cell image of VX765 added after electrotransfer, and FIG. 1C is a cell image of AC-DECD-CHO added after electrotransfer; the scale is 100 microns.

Fig. 2A-2C: to the cell images taken on day 11 of culture of samples of TIL cells double-transferred pS328-PD-1scFv and pS328-PB, fig. 2A is the cell image of the control group (normal electrotransfer culture), fig. 2B is the cell image of VX765 added after electrotransfer, and fig. 2C is the cell image of AC-DECD-CHO added after electrotransfer; the scale is 100 microns.

Fig. 3: cell viability of the different groups on day 3 after electrotransformation.

Fig. 4: cell numbers of different groups on day 3 after electrotransformation.

Fig. 5: cell viability of the different groups on day 11 after electrotransformation.

Fig. 6: cell numbers of different groups on day 11 after electrotransformation.

Fig. 7: cell proliferation profiles of different groups after electrotransformation.

In FIGS. 3-7, CON represents electrotransport TIL without caspase inhibitor added; VX765 represents TIL to which VX765 is added after electrotransformation; AC-DECD-CHO means TIL to which AC-DECD-CHO has been added after electrotransformation.

Fig. 8: and the electric conversion efficiency of the control group.

Fig. 9: after electrotransformation, the electrotransformation efficiency of VX765 is added.

Fig. 10: the electrotransformation efficiency of AC-DECD-CHO was added after electrotransformation.

Detailed Description

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

Caspases are a group of proteases found in the cytoplasm with similar structures, known collectively as cysteine-containing aspartic proteases (CYSTEINYL ASPARTATE SPECIFIC proteinase), which are closely related to eukaryotic apoptosis and are involved in the regulation of cell growth, differentiation and apoptosis. 11 different caspases have been identified and can be divided into 3 subfamilies according to their protease sequence homology: the Caspase-1 subfamily includes Caspase-1, 4,5, 11; the caspase-2 subfamily includes caspase-2, 9; the Caspase-3 subfamily includes Caspase-3, 6, 7, 8, 10.

The invention discovers that after cells, particularly immune effector cells, are transferred into a culture medium for a period of time after the end of electrotransformation, a caspase inhibitor is added, and the caspase inhibitor is any one or more inhibitors of caspase-1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, so that the number and the survival rate of the cells after the electrotransformation can be obviously improved by culturing, thereby completing the invention.

Herein, the cell may be any cell of interest, particularly those conventionally used in the art for electrotransformation to express exogenous 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 may 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. Immune effector cells herein refer to immune cells involved in the clearance of foreign antigens and functioning as an effector 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, 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, a caspase inhibitor may be any of a variety of agents known in the art that inhibit the protease activity of a caspase, including, but not limited to, proteins, nucleic acids, and small molecule compounds that inhibit the protease activity of a caspase. The protein may be, for example, a polypeptide that specifically binds to caspase; the nucleic acid may be an siRNA, antisense RNA, ribozyme, and a gene editing vector, such as a CRISPR-CAS9 gene editing vector or a TALEN gene editing vector; exemplary small molecule compounds include, but are not limited to:

VX765 (caspase-1 inhibitor):

Z-VAD-FMK：

Z-LEHD-FMK (caspase-9 inhibitor):

Z-IETD-FMK (caspase-8 inhibitor):

Z-DEVD-FMK (caspase-3, 6, 7, 8, and 10 inhibitors):

Q-VD-Oph (caspase-1, 3, 8, and 9 inhibitors):

Z-VAD (OH) -FMK (ubiquitin caspase inhibitor):

AC-DEVD-CHO (caspase-3 and-7 inhibitors):

in a preferred embodiment, the caspase inhibitors used in the present invention are inhibitors of the caspase-1 subfamily and the caspase-3 subfamily, more preferably inhibitors of caspase-1 and inhibitors of caspase-3 and/or caspase-7. It is to be understood that certain caspase inhibitors are pan-inhibitors, i.e., can inhibit two or more caspases, and such inhibitors are included within the scope of the present invention, but it is preferred that specific inhibitors of caspase-1 and specific inhibitors of caspase-3 and/or caspase-7 be used, i.e., such inhibitors have an inhibitory effect on even other caspases, but have a much greater inhibitory effect on caspase-1, caspase-3 and/or caspase-7 than on other caspases.

Electrotransformation, also known as electroporation, is used to introduce DNA of interest into host cells. The invention may be practiced using various electrotransport methods and electrotransport reagents well known in the art, e.g., LONZA may be usedI Device and electric transfer reagent provided by the same. The electrotransfer solution can be prepared according to the instruction of the commercial electrotransfer reagent, and the electrotransfer plasmid is added. In electrotransformation, cells are resuspended with an electrotransformation fluid containing an electrotransformation plasmid and electrotransformed in an electrotransformation instrument.

The electrotransport plasmid may be any plasmid of interest, in particular a plasmid expressing a Chimeric Antigen Receptor (CAR). The amount of electrotransport plasmid may be that conventional in the art, for example, 3-8. Mu.g of plasmid is used for electrotransport per 5X 10 ⁶ cells.

After the electrotransformation is completed, the cell suspension is removed, and the pre-heated cell culture medium is added and then cultured under conventional conditions (e.g., 37 ℃, 5% co ₂). After at least 0.5 hour of incubation, caspase inhibitors (especially VX765 and/or AC-DEVD-CHO) are added to the cell culture medium containing electrotransferred cells. Neither too early nor too late addition of caspase inhibitors may increase the total number and viability of the electrotransfer cells. Thus, it is preferred that the caspase inhibitor (especially VX765 and/or AC-DEVD-CHO) is added to the medium within 8 hours, more preferably within 5 hours, more preferably within 3 hours of culturing the electrotransferred cells. For example, in certain particularly preferred embodiments, the caspase inhibitor is added 0.5-5 hours after culturing the electrotransfer cells, preferably 45 minutes to 3 hours after culturing, more preferably 1-2 hours after culturing.

In general, the final concentration of caspase inhibitors (especially VX765 and/or AC-DEVD-CHO) in the post-addition medium is in the range of 5-100. Mu.M, preferably in the range of 10-80. Mu.M, more preferably in the range of 10-50. Mu.M.

Herein, the cell culture medium may be a variety of media suitable for the culture of cells (especially immune cells), especially media conventionally used in the art for the culture of a variety of electrotransformed cells. In certain embodiments, the medium is a medium for culturing immune cells, including but not limited to a medium for culturing one or more of T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. In certain embodiments, the medium is a medium of T cells, TILs, and/or CAR-T cells, particularly a medium of culturing electrotransformed T cells, TILs, and/or CAR-T cells. Exemplary media include, but are not limited toCTS ^TM serum-free cell culture medium, DMEM culture medium, and RPMI1640 culture medium; preferably, it isCTS ^TM serum-free cell culture medium.

After the electrotransfer cells are cultured according to the conventional culture process, compared with the conventional electrotransfer method in the field, the total number of cells and/or the survival rate of the electrotransfer cells can be obviously improved by the culture with the caspase inhibitor.

Accordingly, the present invention provides a method of culturing electrotransformed cells, particularly immune effector cells, comprising adding a caspase inhibitor, particularly VX765 and/or AC-DEVD-CHO, to a culture medium after the electrotransformed cells are transformed into the medium for 0.5-8 hours after termination of the electrotransformation, and continuing the culture. Preferably, the culture is performed by adding a caspase inhibitor (especially VX765 and/or AC-DEVD-CHO) when the culture is performed in the medium for 0.5-5h, 45 min-3 h or 1-2 h.

In certain embodiments, the invention provides a method of electrotransfer for preparing a cell expressing a foreign gene (particularly an immune effector cell), the method comprising:

2) Culturing 1) cells electrotransformed with a foreign gene in a medium containing a caspase inhibitor (especially VX765 and/or AC-DEVD-CHO);

wherein the caspase inhibitor is added after the end of the electrotransformation at 0.5-8h, preferably 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2h of culturing the electrotransformed cells.

The invention also provides the use of caspase inhibitors (especially VX765 and/or AC-DEVD-CHO) in culturing electrotransformed cells, especially immune effector cells.

The invention also provides a cell culture medium comprising one or more caspase inhibitors. The total concentration of caspase inhibitor in the medium is in the range of 5-100. Mu.M, preferably in the range of 10-80. Mu.M. Preferably, the cell culture medium is a medium for immune effector cells. More preferably, the caspase inhibitor is VX765 and/or AC-DEVD-CHO. Thus, in certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells supplemented with VX765 and/or AC-DEVD-CHO, such as 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 conventionally used for culturing T cells, TIL cells, and/or CAR-T cells. More preferably, the medium is a medium conventionally used for culturing electrotransport immune effector cells. VX765 and/or AC-DEVD-CHO are added into the culture medium for culturing the electrotransformed immune effector cells, so that the total number and the survival rate of the cells can be obviously improved. Preferably, the concentration of VX765 and/or AC-DEVD-CHO in the medium is in the range of 5-100. Mu.M, preferably in the range of 10-80. Mu.M. Exemplary immune effector cell culture media of the invention are those containing VX765 and/or AC-DEVD-CHOCTS ^TM serum-free cell culture medium, DMEM culture medium or RPMI1640 culture medium; preferably VX765 and/or AC-DEVD-CHOCTS ^TM serum-free cell culture medium; more preferably VX765 and/or AC-DEVD-CHO containing said concentrationCTS ^TM serum-free cell culture medium.

The invention has the beneficial effects that the death rate of cells after electrotransformation can be obviously reduced and the ratio of the number of living cells to the living cells after the electrotransformation can be improved by using a culture medium containing caspase inhibitors such as VX765 and/or AC-DEVD-CHO to culture the cells after the electrotransformation, especially immune effector cells.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The electrotransport device used in the examples was LONZA Nucleofector ^TM b from Lonza. Antibodies to PD-1 antibodies were purchased from gold, cat No.: a01853-40. streptavidin-PE was purchased from BD under the accession number 554061. Antibodies to the biotin-labeled anti-PD-1 antibodies were prepared by the method conventional in the art from the Johnston company. Other methods and reagents used in the examples are those 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 liver cancer specimen, removing small tissue blocks with size of 1-2mm ³ from different areas of the specimen, and placing one piece in each hole of 24-hole plate. 2mL of complete medium (GT-T551 medium with 10% FBS) and 3000IU/mL of IL-2 were added per well. The 24-well plate was placed in a 37℃and 5% CO ₂ incubator for cultivation. Half-volume changes were made for all wells on days 5-6 after initiation of culture. Then, according to the growth condition of the TIL, half liquid exchange is carried out every 1-2 days. Once the well is full of TIL and all adherent cells have been removed, the TIL within each full well is collected.

Example 2: construction of recombinant expression vectors

The coding sequence (SEQ ID NO: 1) of the PD-1 single chain antibody was synthesized artificially, and the PD-1 single chain antibody was fused with an Fc fragment to form PD-1scFv-Fc. It was installed between EcoRI and SalI cleavage sites of vector pS328 based on the PB transposon system (pS 328 lacks PB transposon sequence compared to pNB 328; pNB328 has the structure and sequence of CN201510638974.7, the entire contents of which are incorporated herein by reference), and the constructed recombinant expression vector was named pS328-PD-1scFv, respectively.

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

Example 3: electrotransformation of pS328-PD-1scFv with pS328-PB TIL (TIL-double plasmid) expressing PD-1scFv was prepared

The TIL-double-turn expressing PD-1scFv was electroturned as follows:

1) Adding AIM-V culture medium into 3 holes in a 12-hole plate in advance, numbering a, b and c, 2mL each hole, and then transferring into a cell incubator to preheat for 1 hour at 37 ℃ with 5% CO ₂;

2) The ratio of the electrotransport liquid with single dosage per hole is carried out according to the following table:

	100μL Nucleocuvette^TM Strip(μL)
		Volume of Nucleofector ^TM solution	82
Electrolysis supplementary solution	18

3) Taking TIL obtained in example 1 to 3 EP tubes, adding 1X 10 ⁷ cells into each EP tube, centrifuging at 1200rpm for 5min, discarding the supernatant, then re-suspending the cells with 500 μl of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) Adding 4 mug of each of the plasmids pS328-PD-1scFv and pS328-PB into the electrotransfer solution prepared in the step 2), and standing at room temperature for less than 30 min;

5) 3 tubes of TIL are resuspended by using the plasmid-containing electrotransfer solution prepared in 4), 100 mu L of each tube is carefully sucked up and transferred into a LONZA 100 mu L electrotransfer cup, the electrotransfer cup is placed into a LONZANucleofector ^TM b electrotransfer tank, an electrotransfer program is started, and the electrotransfer program selects X001;

6) Carefully taking out an electric rotating cup after electric rotating, sucking cell suspension, transferring the cell suspension into an EP tube, adding 200 mu L of preheated AIM-V culture medium into each tube, and then transferring the cell suspension into three holes a, b and c of a 12-well plate containing the preheated AIM-V culture medium in 1), and culturing at 37 ℃ with 5% CO ₂;

7) VX765 and AC-DECD-CHO were added to the a-well and the b-well, respectively, at a final concentration of 50. Mu.M at the time point of 1h of incubation; well c was not added with caspase inhibitor and was used as a control well, followed by continued culture.

The culturing operation was repeated 3 times, and the total number of cells and the percentage of living cells in each well after culturing for 3 and 11 days were counted, respectively, and the average was taken.

The results are shown in FIGS. 1-7.

FIGS. 1A-1C show that samples of double-transferred pS328-PD-1scFv and pS328-PB showed significantly more TIL in the VX765 and AC-DECD-CHO groups than in the control cells without caspase inhibitor at day 6 of culture. FIGS. 2A-2C show that samples of the double-turn pS328-PD-1scFv and pS328-PB on day 11 of culture had significantly more TIL in the VX765 group and the AC-DECD-CHO group than in the control group without caspase inhibitor.

FIG. 3 shows that the ratio of living cells of VX765 group to AC-DECD-CHO group was significantly higher than that of control group after 3 days of electrotransformation. FIG. 4 shows that the total number of cells in VX765 group and AC-DECD-CHO group was greater than that in control group after 3 days of electrotransformation.

FIG. 5 shows that the ratio of living cells of VX765 group to that of AC-DECD-CHO group was significantly higher than that of control group after 11 days of electrotransformation. FIG. 6 shows that the total number of cells in VX765 group and AC-DECD-CHO group was greater than that in control group after 11 days of electrotransformation.

FIG. 7 shows that the proliferation level of VX765 group and AC-DECD-CHO group was significantly higher than that of the control group, and that the proliferation level of AC-DECD-CHO group was slightly higher than that of VX765 group, as compared with the control group.

The results in FIGS. 1-7 show that caspase inhibitors significantly increase the cell activity and survival of TILs following electrotransport plasmids.

VX765 and AC-DECD-CHO were added to final concentrations of 10. Mu.M or 80. Mu.M at time points of 0.5h or 5h of incubation in step 7) to obtain results similar to FIGS. 1-7.

Example 4: flow cytometer for detecting electrotransformation efficiency of each group of cells

Cells from the control group (CON), VX765 group and AC-DECD-CHO group were examined by flow cytometry for each of TIL double-transferred pS328-PD-1scFv and pS328-PB plasmid following 12 days of culture, and PD-1scFv expression levels were examined as in example 3. The specific operation is as follows:

1. the biotin-labeled anti-PD-1 antibody is dissolved in PBS to prepare working solution with the concentration of 1 mg/mL;

2. taking 1× ⁶ cells of a control group (CON), VX765 group and AC-DECD-CHO group respectively, centrifuging at 1000rpm for 3min, discarding the upper culture medium, adding 2 μl of biotin-labeled anti-PD-1 antibody working solution, and incubating at 37deg.C and 5% CO ₂ for 1 hr;

3. The cells of each group after incubation in step 2 were washed three times with cold PBS, resuspended in 100. Mu.L of physiological saline, and incubated with 1. Mu.L of streptavidin-PE at 4℃for 30 minutes. After washing three times with physiological saline, the fluorescence intensity of the cells was measured by a flow cytometer, and the positive rate of each group of cells was analyzed. The results are shown in FIGS. 8-10.

FIGS. 8, 9 and 10 show that the proportion of PD-1scFv expressing positive cells in the control, VX765 and AC-DECD-CHO TILs was 22.7%, 23.73% and 20.40%, respectively, and that the proportion of PD-1 antibody expressing positive cells in the three groups of cells was substantially equivalent, indicating that the use of caspase inhibitor small molecules in electrotransport TILs had little effect on transfection efficiency.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above description of the application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai cell therapy group Co., ltd

Shanghai cell therapy institute

<120> Method for preparing cells overexpressing foreign genes

<130> 18A449

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1488

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180

caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240

ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300

agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360

acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420

agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480

ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540

tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600

aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660

tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720

tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780

accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840

gagttcgagg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt tccagagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488

Claims

Use of a caspase inhibitor in the culture of electrotransport cells, said caspase inhibitor being:

；

the cells are TIL cells.
2. A method of culturing electrotransformed cells, the method comprising the step of culturing the electrotransformed cells in a medium comprising a caspase inhibitor:

；

the cells are TIL cells.
3. The method of claim 2, comprising, after the electrotransformation is completed, transferring the electrotransformed cells into a culture medium for 0.5-5 hours, and then adding the caspase inhibitor to the culture medium to continue the culture.
4. The method of claim 3, wherein the caspase inhibitor is added at a final concentration in the medium in the range of 5-100 μm.
5. The method of claim 3, wherein the caspase inhibitor is added at a final concentration in the medium in the range of 10-80 μm.
6. The method of claim 2, wherein the medium is a cell culture medium.
7. The method of claim 6, wherein the medium is a medium for culturing immune effector cells.
8. The method of claim 7, wherein the medium is selected from the group consisting of GIBCO-AIM-V CTS ™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.
9. The method of claim 8, wherein the medium is a GIBCO cube AIM-V CTS ™ serum-free cell culture medium.
10. A method for preparing a cell expressing a foreign gene by electrotransformation, the method comprising:

1) Introducing a nucleic acid comprising an expressed exogenous gene into the cell by electrosteering;

2) Culturing the cells having the exogenous gene electrotransferred in step 1) with a medium containing a caspase inhibitor;

Wherein the electroporated cells are cultured for 0.5-5 hours, and then the caspase inhibitor is added to the culture medium,

The caspase inhibitor is:

；

the cells are TIL cells.
11. The method of claim 10, wherein the medium is a medium for culturing immune effector cells.
12. The method of claim 11, wherein the medium is selected from the group consisting of GIBCO-AIM-V CTS ™ serum-free cell culture medium, DMEM medium, and RPMI1640 medium.
13. The method of claim 12, wherein the medium is a GIBCO cube-V CTS ™ serum-free cell culture medium.