CN111349615A

CN111349615A - Method for preparing cell over expressing exogenous gene

Info

Publication number: CN111349615A
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: 2020-06-30
Anticipated expiration: 2038-12-24
Also published as: CN111349615B

Abstract

The present invention relates to a method for producing a cell overexpressing a foreign gene. The invention utilizes caspase inhibitor to culture electrotransfer cell, especially electrotransfer immune effector cell, and can obviously improve the survival rate and total cell number of the cell after 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 mainly involves the use of retroviral or lentiviral vectors to express the CAR gene, the introduction of the CAR gene into immune effector cells by packaged viral particles, and integration into the cell genome by the retroviral or lentiviral self-integrating 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 accessible by several common electrotransfer procedures. In electroporation-based transgenic procedures, exogenous DNA is introduced intracellularly by reversible electroporation, and the exogenous gene is expressed in its new host cell and inherited as the cell divides. The use of the method of electric transfer in combination with a non-viral gene modification system capable of inducing stable expression of transgenes, such as the 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 Cell to automatic Disease Control in Patients under automated Autologus telematics step-Cell transformation for Advanced B-Cell amplification in T cells. human therapy 23: 444-450), which have higher efficiency in T cells. ACT using a transposon system relies on electrotransformation of T cells and Tumor Infiltrating Lymphocytes (TIL), for which there are currently well established commercial electrotransformation machines and buffers (e.g., Lonza Nuclear organisms), recently there are also reports of methods for successfully constructing CAR-T cells by commercial electrotransformation systems using SB transposon systems (Jin Z, MaitiS, Huls H, Singh H, Olivares S, et al (2011) The genetic modification of T cells to expression vectors, Gene Therapy 18: 849;. PenPD, Cohen, Yang S, C, Joule S2009, Biogene Therapy methods 16, and The genetic transformation methods of PCR-T cells (Japanese patent application Biogene et 4: CJ PD, genetic engineering, Yang S, Japan, C, Job et al) are used for genetic transformation of Tumor Infiltrating lymphocytes (T cells) and The genetic transformation methods of PCR-11 strain Escherichia coli cells (Japanese patent application Biogene et 4: S) are used in The genetic testing of transgenic cells by The transgenic animal assay of PCR-mediated transformation system, huls H, Jena B, Munsell M, Jackson R, et al (2012) Infusng CD19-Directed T Cells to automatic distance Control in Patients Underg Autologous animal Stem-Cell transfer for Advanced B-Circuit Malignanceae. human Gene Therapy 23: 444-.

Compared with a viral vector system, the electrotransport and transposon 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). Particularly, the difficulty of electrotransformation of immune effector cells, such as PBMC and TIL cells, is relatively higher, and although mature electrotransformation instruments and matched buffer systems are already available in 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

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

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

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 present invention also provides a method of culturing an electroporated cell, the method comprising the step of culturing the electroporated cell 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 electrotransformation is finished, transferring the electrotransformed cells into a culture medium for 0.5 to 5 hours, then adding the caspase inhibitor to the culture medium, and continuing the culture.

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 a preferred embodiment, 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.

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 culture medium is a cell culture medium, preferably a medium for culturing immune effector cells; preferably selected from

CTS^TMSerum-free cell culture medium, DMEM medium and RPMI1640 medium; more preferably

CTS^TMSerum-free cell culture media.

The present invention also provides a method for preparing a cell expressing an exogenous 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 with the exogenous genes electrically transferred in the step 1) by using a culture medium containing a caspase preparation;

wherein the caspase inhibitor is added into the culture medium after the electrotransferred cells are cultured for 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2h, and the culture 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 from

CTS^TMSerum-free cell culture media.

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

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

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

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 from

CTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; more preferably

CTS^TMSerum-free cell culture media.

Drawings

FIGS. 1A-1C: to TIL cells, images of cells taken on day 6 of culture were generated by double-transferring samples of pS328-PD-1scFv and pS 328-PB. FIG. 1A is an image of cells in a control group (normal electroporation culture), FIG. 1B is an image of cells after electroporation to which VX765 was added, and FIG. 1C is an image of cells after electroporation to which AC-DECD-CHO was added; the scale is 100 microns.

FIGS. 2A-2C: to TIL cells, images of cells taken on day 11 of culture for samples of double-transfected pS328-PD-1scFv and pS328-PB, FIG. 2A is an image of cells of a control group (normal electroporation culture), FIG. 2B is an image of cells after electroporation to which VX765 was added, and FIG. 2C is an image of cells after electroporation to which AC-DECD-CHO was added; the scale is 100 microns.

FIG. 3: viability of different groups of cells on day 3 of culture after electroporation.

FIG. 4: the number of cells of different groups was cultured on day 3 after electroporation.

FIG. 5: viability of different groups of cells on day 11 of culture after electroporation.

FIG. 6: the number of cells of different groups was cultured on day 11 after electroporation.

FIG. 7: cell proliferation change curves of different groups after electrotransformation.

In FIGS. 3-7, CON represents electrotransfer TIL without caspase inhibitor added; VX765 represents a TIL to which VX765 was added after electrotransfer; AC-DECD-CHO represents TIL to which AC-DECD-CHO was added after electrotransformation.

FIG. 8: electrotransfer efficiency of control group.

FIG. 9: and adding VX765 after electric conversion.

FIG. 10: and adding AC-DECD-CHO after electric conversion to obtain the electric conversion efficiency.

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 described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

Caspases are a group of proteases with similar structures existing in cytoplasm and are all called cysteine-containing aspartic acid proteolytic enzymes (cysteine aspartic acid proteolytic enzymes), are closely related to the apoptosis of eukaryotic cells and are involved in the growth, differentiation and apoptosis regulation of cells. 11 different caspases have been identified and classified into 3 subfamilies based on 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 electrically transferred to a culture medium and cultured for a period of time, caspase inhibitors which are any one or more of caspase-1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 are added to the cells, and the number and the survival rate of the cells after the cells are electrically transferred can be obviously improved by culturing the cells, thereby completing the invention.

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, caspase inhibitors 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 a caspase; the nucleic acid can be siRNA, antisense RNA, ribozymes, and gene editing vectors, such as CRISPR-CAS9 gene editing vectors or TALEN gene editing vectors; 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 (pan-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 and caspase-3 subfamilies, more preferably inhibitors of caspase-1 and inhibitors of caspase-3 and/or caspase-7. It will be appreciated that certain caspase inhibitors are pan inhibitors, i.e. inhibit two or more caspases, and such inhibitors are also included within the scope of the present invention, but it is preferred to use specific inhibitors of caspase-1 and specific inhibitors of caspase-3 and/or caspase-7, i.e. such inhibitors have an inhibitory effect on caspase-1, caspase-3 and/or caspase-7 which is much stronger than the inhibitory effect on other caspases, even though they also inhibit other caspases.

Electrotransfer is also referred to as electroporation,for introducing the DNA of interest into the host cell. The invention can be practiced using various electrotransformation methods and agents well 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 electrotransformation plasmid may be any plasmid of interest, in particular a plasmid expressing a Chimeric Antigen Receptor (CAR). the amount of electrotransformation plasmid may be an amount conventional in the art, for example every 5 × 10⁶Each cell was electroporated with 3-8. mu.g of plasmid.

After the electrotransfer is complete, the cell suspension is removed, preheated cell culture medium is added, and the mixture is incubated under conventional conditions (e.g., 37 ℃ C., 5% CO)₂) And (5) culturing. After at least 0.5 hour of culture, caspase inhibitors (especially VX765 and/or AC-DEVD-CHO) are added to the cell culture medium containing the transfected cells. Either premature or late addition of caspase inhibitors may fail to increase the total number and survival of electroporated cells. Therefore, it is preferred that a caspase inhibitor (especially VX765 and/or AC-DEVD-CHO) is added to the culture medium within 8 hours, more preferably within 5 hours, more preferably within 3 hours of culturing the electroporated cells. For example, in certain particularly preferred embodiments, the caspase inhibitor is added when the electroporated cells are cultured for 0.5-5h, preferably 45min to 3h, and more preferably 1-2 h.

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 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 for culturing immune cellsCell culture media, including but not limited to media 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 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^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, is

CTS^TMSerum-free cell culture media.

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

Accordingly, the present invention provides a method for culturing an electrotransferred cell, particularly an immune effector cell, which comprises adding a caspase inhibitor, particularly VX765 and/or AC-DEVD-CHO, to a culture medium after the electrotransferred cell is transferred to the culture medium and cultured for 0.5 to 8 hours after the end of the electrotransferred cell, and continuing the culture. Preferably, caspase inhibitors (especially VX765 and/or AC-DEVD-CHO) are added for culture at 0.5-5h, 45 min-3 h or 1-2h in the culture medium.

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

2) culturing the cells having the foreign gene transferred thereto in a medium containing a caspase inhibitor (particularly VX765 and/or AC-DEVD-CHO) of 1);

wherein the caspase inhibitor is added after the electrotransfer is finished and when the electrotransfer 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 a caspase inhibitor, particularly VX765 and/or AC-DEVD-CHO, in culturing transfected cells, particularly 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, 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, and in particular a medium conventionally used for culturing T cells, TIL cells, and/or CAR-T cells, to which VX765 and/or AC-DEVD-CHO is added. More preferably, the medium is a medium conventionally used for culturing electroporated immune effector cells. The addition of VX765 and/or AC-DEVD-CHO to such a medium for culturing the transfected immune effector cells can significantly improve the total number and survival rate of the cells. 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-CHO

CTS^TMSerum-free cell culture medium, DMEM medium or RPMI1640 medium; preferably, the modified starch is VX765 and/or AC-DEVD-CHO

CTS^TMSerum-free cell culture media; more preferably VX765 and/or AC-DEVD-CHO at said concentrations

CTS^TMSerum-freeCell 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 using the culture medium containing caspase inhibitors, such as VX765 and/or AC-DEVD-CHO to culture the cells after the electrotransformation, in particular immune effector cells.

The present invention will be illustrated below 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 present invention. The electrotransformation instrument used in the examples was a LONZA Nucleofector from Lonza^TM2b, and 2 b. Antibodies to PD-1 antibodies were purchased from kasugar, cat #: a01853-40. streptavidin-PE was purchased from BD, cat # 554061. An antibody against PD-1 antibody labeled with biotin was prepared by a method conventional in the art by Kinsley. 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 specimen³One for each well of a 24-well plate. Add 2mL of complete medium (GT-T551 medium with 10% FBS) and 3000IU/mL IL-2 per well. The 24-well plate was placed at 37 ℃ in 5% CO₂Culturing 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 coding sequence (SEQ ID NO: 1) of the PD-1 single-chain antibody was artificially synthesized, and the PD-1 single-chain antibody was fused with an Fc fragment to form PD-1 scFv-Fc. The recombinant expression vectors constructed were placed between EcoRI and SalI cleavage sites of the PB transposon system based vector pS328 (pS328 lacks the PB transposon sequence compared to pNB 328; the structure and sequence of pNB328 is referred to as CN201510638974.7, which is herein incorporated by reference in its entirety), and were designated pS328-PD-1scFv, 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 as SEQ ID NO:5 in CN201510638974.7, and the constructed recombinant expression vector was named pS 328-PB.

Example 3: electrotransfer of pS328-PD-1scFv and pS328-PB TIL expressing PD-1scFv (TIL-Dual plasmid)

Electrically transduce TIL-bi-transgenes for PD-1scFv as follows:

1) AIM-V medium was added to 3 wells of 12-well plates, numbered a, b, c, 2mL per well in advance, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating 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^TMvolume of solution	82
Electrotransformation make-up solution	18

3) The TIL obtained in example 1 was taken out into 3 EP tubes, and 1 × 10 was added to each EP tube⁷Centrifuging 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 plasmids pS328-PD-1scFv and pS328-PB into the electrotransfer solution prepared in the step 2) to 4 mu g of each, and then standing at room temperature for less than 30 min;

5) resuspending 3 tubes of TIL with 100. mu.L of plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects X001;

6) after the completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to three wells a, b, and c containing preheated AIM-V medium in 12-well plate of 1), 37 ℃ and 5% CO₂Culturing;

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

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

The results are shown in FIGS. 1 to 7.

FIGS. 1A-1C show that the amount of TIL in the VX765 and AC-DECD-CHO groups was significantly greater than that in the control group to which no caspase inhibitor was added, for the samples of the dual-rotor pS328-PD-1scFv and pS328-PB at day 6 of culture. FIGS. 2A-2C show that the amount of TIL in the VX765 and AC-DECD-CHO groups was significantly greater for the samples of dual-rotor pS328-PD-1scFv versus pS328-PB at day 11 of culture than for the control group without caspase inhibitor.

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

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

FIG. 7 shows that compared to the control group, the proliferation levels of the VX765 group and the AC-DECD-CHO group are significantly higher than the control group, and the proliferation level of the AC-DECD-CHO group is slightly higher than that of the VX765 group.

The results of FIGS. 1-7 show that caspase inhibitors can significantly improve the cell activity and survival rate of TIL after plasmid electrotransformation.

Step 7) VX765 and AC-DECD-CHO were added at the time point of 0.5h or 5h of culture to a final concentration of 10. mu.M or 80. mu.M, and results similar to those of FIGS. 1-7 were obtained.

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

The expression level of PD-1scFv was measured in example 3 by flow cytometry on cells of control group (CON), VX765 and AC-DECD-CHO, respectively, after 12 days of culture on the TIL double-rotor pS328-PD-1scFv and pS328-PB plasmids. The specific operation is as follows:

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

2. cells from Control (CON), VX765 and AC-DECD-CHO groups were each 1 × 10⁶Centrifuging at 1000rpm for 3min, discarding the upper culture medium, adding 2 μ L biotin-labeled anti-PD-1 antibody working solution, 37 deg.C, and 5% CO₂Incubating for 1 h;

3. the incubated groups of cells from step 2 were washed three times with cold PBS, resuspended in 100. mu.L of physiological saline, added with 1. mu.L of streptavidin-PE and incubated at 4 ℃ for 30 minutes. After three times of washing with physiological saline, the fluorescence intensity of the cells was measured by flow cytometry, 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 expression positive cells in TILs of control, VX765 and AC-DECD-CHO groups was 22.7%, 23.73% and 20.40%, respectively, and that the proportion of PD-1 antibody expression positive cells in TILs of the three groups was substantially equivalent, indicating that the use of caspase inhibitor small molecules in the electrotransfer TILs had little effect on transfection efficiency.

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 and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shanghai cell therapy group Co., Ltd

SHANGHAI CELL THERAPY Research Institute

<120> method for preparing cell overexpressing foreign gene

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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

The application of caspase inhibitor in culturing electrically transferred cell;

preferably, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitors, caspase-2 inhibitors, caspase-3 inhibitors, caspase-4 inhibitors, caspase-5 inhibitors, caspase-6 inhibitors, caspase-7 inhibitors, caspase-8 inhibitors, and caspase-10 inhibitors; more preferably, the caspase inhibitor is a caspase-1 inhibitor, a caspase-3 inhibitor or a caspase-7 inhibitor; more preferably, the caspase inhibitor is:

preferably, the cell is an immune effector cell.
2. A method of culturing an electroporated cell, comprising the step of culturing the electroporated cell in a medium comprising a caspase inhibitor;

preferably, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitors, caspase-2 inhibitors, caspase-3 inhibitors, caspase-4 inhibitors, caspase-5 inhibitors, caspase-6 inhibitors, caspase-7 inhibitors, caspase-8 inhibitors, and caspase-10 inhibitors; more preferably, the caspase inhibitor is a caspase-1 inhibitor, a caspase-3 inhibitor or a caspase-7 inhibitor; more preferably, the caspase inhibitor is:

preferably, the cell is an immune effector cell.
3. The method according to claim 2, comprising, after the completion of the electrotransformation, transferring the electrotransformed cells to a culture medium and culturing for 0.5 to 5 hours, and then adding the caspase inhibitor to the culture medium and continuing the culturing; preferably, 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.
4. The method of claim 2, wherein the immune effector cells are selected from one or more of T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; preferably selected from one or more of T cells, TILs and CAR-T cells.
5. As claimed in claim 2The method is characterized in that the culture medium is a cell culture medium, preferably a culture medium for culturing immune effector cells; preferably selected from
CTS^TMSerum-free cell culture medium, DMEM medium and RPMI1640 medium; more preferably
CTS^TMSerum-free cell culture media.
6. 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 with the exogenous genes electrically transferred in the step 1) by using a culture medium containing a caspase preparation;

wherein the electroporated cells are cultured for 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2h, and then the caspase inhibitor is added to the culture medium; preferably, the final concentration of the caspase inhibitor in the culture medium after addition is in the range of 5-100. mu.M, preferably 10-80. mu.M;

preferably, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitors, caspase-2 inhibitors, caspase-3 inhibitors, caspase-4 inhibitors, caspase-5 inhibitors, caspase-6 inhibitors, caspase-7 inhibitors, caspase-8 inhibitors, and caspase-10 inhibitors; more preferably, the caspase inhibitor is a caspase-1 inhibitor, a caspase-3 inhibitor or a caspase-7 inhibitor; more preferably, the caspase inhibitor is:

preferably, the cell is an immune effector cell.
7. The method of claim 6, 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; preferably selected from T cells, TILs and CAR-T cells.
8. The method of claim 6, wherein the medium is a medium for culturing immune effector cells; preferably selected from
CTS^TMSerum-free cell culture medium, DMEM medium and RPMI1640 medium; more preferably
CTS^TMSerum-free cell culture media.
9. A cell culture medium, wherein said cell culture medium is supplemented with a caspase inhibitor;

preferably, the concentration of caspase inhibitor in said cell culture medium is 5-100. mu.M, preferably 10-80. mu.M;

preferably, the caspase inhibitor is selected from the group consisting of caspase-1 inhibitors, caspase-2 inhibitors, caspase-3 inhibitors, caspase-4 inhibitors, caspase-5 inhibitors, caspase-6 inhibitors, caspase-7 inhibitors, caspase-8 inhibitors, and caspase-10 inhibitors; more preferably, the caspase inhibitor is a caspase-1 inhibitor, a caspase-3 inhibitor or a caspase-7 inhibitor; more preferably, the caspase inhibitor is:
10. the cell culture medium of claim 9, wherein the cell culture medium is for culturing an immune responseThe medium of the cells is preferably selected from
CTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; more preferably
CTS^TMSerum-free cell culture media.