CN106957822B - Culture method, kit and application of in-vitro amplified gene editing activated T cells - Google Patents
Culture method, kit and application of in-vitro amplified gene editing activated T cells Download PDFInfo
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Abstract
The invention provides a culture medium for in vitro amplification of gene editing activated T cells, which is characterized by comprising a T cell basic culture medium and further comprising: interleukin 7(IL-7)200-1000IU/ml, interleukin 21(IL-21)300-1000IU/ml, anti-CD 40 monoclonal antibody 80-120ng/ml, serine 0.2-1.0mmol/ml and S-2-hydroxyglutaric acid 0.3-1.0 mmol/ml. The invention also provides a kit containing the culture medium, a method for amplifying the gene editing and activating T cells in vitro by using the culture medium and application in the aspect of culturing the T cells. The culture medium of the invention can be used for amplifying a large number of gene editing activated T cells without adding allogenic serum and other activated T cell factors. The method can activate and amplify T cells by 500-fold (1000-fold), which is obviously higher than the prior art.
Description
Technical Field
The invention belongs to the technical field of biotechnology and cell culture, and particularly relates to a culture method for in vitro amplification gene editing activated T cells, a kit and application thereof. The method comprises but is not limited to a functional activated T cell culture method constructed by adopting genome editing technologies such as ZFN, TALEN, CRISPR-CAS9 and the like, a kit and application thereof.
Background
With the continuous and deep immunological research, the tumor immunotherapy has made great progress. Tumor immunotherapy is also considered as the fourth major tumor treatment technology after surgery, radiotherapy and chemotherapy, and can enhance the anti-tumor immunity of tumor microenvironment by stimulating or mobilizing the patient's own immune system, thereby controlling and killing tumor cells. In the last decade, tumor immunotherapy methods represented by Adoptive Cell Therapy (ACT) and immune checkpoint therapy (immune checkpoint therapy) have made breakthrough progress, show good application prospects, and mark the opening of a new era of tumor therapy.
In tumor immunotherapy protocols, T lymphocytes are in a central position. The low immune function of tumor-specific cells in vivo is one of the important reasons for tumors to escape the immune system's surveillance of unrestricted growth. The cell biotherapy technology is to induce, differentiate and amplify the autoimmune cells in vitro and then return the autoimmune cells to the body, bypass the tumor immune barrier mechanism in the body, and resist, inhibit and kill the cancer cells by exciting the immune response of the body. How to obtain a sufficient amount of T cells for clinical application in vitro is a primary problem in adoptive immunotherapy of tumors.
Genome editing (Genome editing) is a genetic manipulation technology for site-directed modification of DNA sequences at the Genome level, and is known as a booster for life science research in the post-Genome era. The principle of genome editing technology is to cut off DNA at a target site by constructing an artificial endonuclease, generate DNA Double Strand Breaks (DSB), and further induce the intracellular DNA repair system to perform nonhomologous end joining (NHEJ) and Homologous recombination repair (HR). Through the two repair approaches, the genome editing technology can realize site-specific gene knockout, specific mutation introduction and site-specific modification. Until genome editing techniques were not discovered, scientists could only target genome modification by homologous recombination. However, the method excessively depends on natural recombination, has extremely low efficiency and cannot meet the increasingly developed scientific research requirements of human beings. In this case, gene editing techniques have been developed. In recent years, gene editing has become simple and feasible due to the rise of the technology of artificial endonuclease (EEN), and the EEN technology which is currently used is mainly Zinc Finger Nuclease (ZFN), transcription activator-like nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR).
At present, in tumor cell immunotherapy, T cells are the core role of anti-tumor cell immunity, however, tumor cells often express some ligands to bind with T cells, and then inhibit activation, proliferation and killing of T cells on tumors, so that the functions of T cells are disordered and exhausted, thereby achieving the purpose of immune evasion. The specific gene or gene group in the T cell is knocked out by a gene editing technology, so that the purpose of killing tumor cells can be better achieved.
Disclosure of Invention
The invention aims to improve the amplification speed of T cells treated by a gene editing technology, reduce the amplification time, provide a culture method, a culture medium and application thereof, which can economically, simply and efficiently amplify gene editing activated T cells, and further improve the therapeutic effect of the gene editing activated T cells on tumors or other diseases.
The invention provides a culture medium for in vitro amplification of gene editing activated T cells, which comprises a T cell basic culture medium and also comprises: interleukin 7(IL-7)200-1000IU/ml, interleukin 21(IL-21)300-1000IU/ml, anti-CD 40 monoclonal antibody 80-120ng/ml, serine 0.2-1.0mmol/ml and S-2-hydroxyglutaric acid 0.3-1.0 mmol/ml.
Preferably, the culture medium comprises a T cell basal medium, further comprising: 500IU/ml of interleukin 7(IL-7), 550IU/ml of interleukin 21(IL-21), 100ng/ml of anti-CD 40 monoclonal antibody, 0.45mmol/ml of serine and 0.5mmol/ml of S-2-hydroxyglutaric acid.
Preferably, the basic culture medium is RPMI-1640 culture medium.
The culture medium of the present invention can be used not only for culturing gene-editing activated T cells but also for culturing ordinary T cells.
It is therefore a further object of the invention to provide the use of said medium for the cultivation of T-cells.
The invention provides a method for amplifying gene editing activated T cells in vitro, which comprises the following steps:
(1) pre-culturing the T cells after gene modification, wherein the culture medium comprises a basal culture medium and 80-120ng/ml of anti-CD 40 monoclonal antibody;
(2) after pre-culture, adding 1000IU/ml of interleukin 7(IL-7) 200-.
Preferably, in step (1), the basal medium is RPMI-1640 medium.
Preferably, in step (1), the culture time is 48 hours.
Preferably, in the step (2), 500IU/ml of interleukin 7(IL-7), 550IU/ml of interleukin 21(IL-21), 100ng/ml of anti-CD 40 monoclonal antibody, 0.45mmol/ml of serine and 0.5mmol/ml of S-2-hydroxyglutaric acid are added into the culture medium in the step (1) to perform high-efficiency amplification culture.
Alternatively, the genetic modification technique comprises ZEN, TALE N or CRISPR/Cas 9.
The genetic modification is carried out by modifying the relevant activating and/or repressing genes in the T-cell.
CD40 is one of T cell surface membrane protein receptors, CD40 can be combined with a ligand thereof to play a role of PD-1 antagonist by activating mTORC1 pathway, so that the effect of promoting the rapid proliferation of T cells is achieved, and the anti-CD 40 antibody is combined with CD40 to activate mTORC1 pathway, so that the rapid proliferation of T cells is promoted.
Serine and S-2-hydroxyglutaric acid added into the culture medium are all substances necessary for energy metabolism of the T cells in the rapid proliferation process, and the proliferation speed of the T cells can be greatly improved by additionally adding a certain amount of serine and S-2-hydroxyglutaric acid.
The culture medium can be used for amplifying a large number of gene editing activated T cells under the condition of not adding allogenic serum and other activated T cell factors, and the induction method of the culture medium is quick, efficient, economical, simple and stable in effect. In addition, the detection result of the flow cytometer commonly used in the industry indicates that the method for amplifying the gene editing and activating the T cells in vitro can activate and amplify the T cells by 500-fold and 1000-fold, which is obviously higher than that of the prior art.
Detailed Description
The following examples are further illustrative of the present invention but the present invention is not limited to these specific embodiments. The gene editing technology used in the gene editing activated T cell mainly comprises ZFN, TALEN, CRISPR-Cas9 and the like, but the invention is not limited to the use of the three gene editing technologies. The culture method, the kit and the application of the in vitro amplified gene editing activated T cell are mainly used for T cell in vitro separation, induction, differentiation, culture, amplification and the like; the processed biological products comprise human peripheral blood, cord blood, pleural effusion, ascites and the like; can be applied to clinical cell biological treatment, including malignant tumor diseases such as lung cancer, kidney cancer, melanoma, leukemia, breast cancer, rectal cancer, stomach cancer, esophagus cancer, cervical cancer, ovarian cancer, bone marrow cancer, malignant lymphoma and the like, can also be applied to basic medical research, clinical application research and biotechnology research, and particularly can be applied to immune system research and tumor killing effect research. The specific embodiment is as follows:
example 1 (Experimental group)
Separation and high-efficiency amplification culture of human PD-1 gene T cell knocked out by CRISPR-Cas9 technology
Human peripheral blood is taken as a treatment sample, a human PD-1 gene is knocked out by using a CRISPR-Cas9 technology, a PD-1 gene knockout T cell is constructed, and the gene editing and activating T cell is subjected to in vitro high-efficiency amplification culture. The method comprises the following specific steps:
the method comprises the following steps: separation of peripheral blood mononuclear cells from blood
Directly extracting 80-100 ml of donor venous blood, and adding an anticoagulant heparin;
centrifuging the collected blood sample at room temperature at 2000rpm for 10min, and carefully sucking the upper plasma layer for culture; reducing the blood sample to the original volume, and mixing uniformly; slowly adding diluted blood onto Ficoll, and centrifuging at 1000rpm for 20 min; sucking the milky mononuclear cell layer on the interface of the separation solution, and centrifugally washing for 2 times to obtain PBMC.
Step two: isolation of T cells from PBMC
Taking separated PBMC, adding basal medium RPMI-1640 to prepare cell suspensionAdjusting the cell concentration to 1-2 × 106Per ml, 37 ℃, 5% CO2After 2 hours of incubation, the suspension cells were collected as T cells.
Step three: human PD-1 gene on T cell knocked out by CRISPR-Cas9 technology
Construction of sgRNA oligonucleotide viral vector carrying PD-1 gene
According to the design principle of sgRNA, designing a target sequence of the sgRNA on the PD-1 gene, synthesizing a sgRNA oligonucleotide double strand, annealing at 95 ℃ for 5min after mixing, and then connecting with a linearized lentiviral vector to obtain a sgRNA oligonucleotide viral vector carrying the PD-1 gene;
construction of vectors carrying Cas 9-nuclease related sequences
Cloning a Cas9 nuclease into a related vector to obtain a Cas 9-nuclease vector;
transduction of T cells
Synchronously transducing the T cells collected in the second step with corresponding sgRNA and Cas 9-nuclease in a specific mode to knock out the human PD-1 gene, namely obtaining the PD-1 gene-knocked-out T cells.
Step four: pre-cultured PD-1 gene knockout T cells
Collecting PD-1 gene knockout T cells, suspending and culturing in a basal medium RPMI-1640, adding 80-120ng/ml of anti-CD 40 antibody, 37 ℃, and 5% CO2And (5) incubating and culturing for 48 h.
Step five: efficient amplification culture of PD-1 gene knockout T cells
Adding 500IU/ml of interleukin 7(IL-7), 550IU/ml of interleukin 21(IL-21), 100ng/ml of anti-CD 40 monoclonal antibody, 0.45mmol/ml of serine and 0.5mmol/ml of S-2-hydroxyglutaric acid into the T cell culture solution cultured for 48 hours in the fourth step, continuing culturing, half-replacing the culture solution every two days, wherein the half-replacing solution is a culture medium taking half of cells, removing the culture medium after centrifugally collecting the cells, adding RPMI-1640 culture medium containing 500IU/ml of interleukin 7(IL-7), 550IU/ml of interleukin 21(IL-21), 100ng/ml of anti-CD 40 monoclonal antibody, 0.45mmol/ml of serine and 0.5mmol/ml of S-2-hydroxyglutaric acid, and adjusting the cell concentration to be 1 × 106One per ml.
After culturing for 48h from the fifth step, the T cells were transferred to T175 flasks for further culturing. To ensure sufficient growth space for the cells.
And D, culturing for 14-21 days from the fifth step, collecting the T cells, centrifuging the T cells, and washing the T cells for three times by using physiological saline to obtain the activated T cells.
Comparative example 1: no anti-CD 40 monoclonal antibody, serine, S-2-hydroxyglutaric acid (control 1)
The procedure was as in example 1 except that the anti-CD 40 monoclonal antibody, serine, and S-2-hydroxyglutaric acid were not added in step four and step five of example 1.
Comparative example 2 (control 2):
the procedure was as in example 1, except that the anti-CD 40 monoclonal antibody was added in step four and step five of example 1 at a concentration of 70ng/ml and serine was added at a concentration of 1.1 mmol/ml.
COMPARATIVE EXAMPLE 3 (COMPARATIVE GROUP 3)
The procedure was as in example 1, except that the anti-CD 40 monoclonal antibody was added in step four and step five of example 1 at a concentration of 130ng/ml and S-2-hydroxyglutaric acid at a concentration of 0.25 mmol/ml. The gene-edited T cells obtained in the experimental group and the control groups 1 to 3 of example 1 were subjected to fold amplification detection.
On days 1, 7, 14 and 21, 200. mu.l of the cells were aspirated into single cells, and the cells were counted using a Counterstar automatic hemocytometer (Counterstar Autocytometer), and the cell expansion factor was the total number of cells counted on the same day/the total number of cells before culture on day 1. The results are shown in Table 1.
TABLE 1 variation of cell number of T cells at different times (× 10)8/L)
Claims (8)
1. A culture medium for in vitro amplification of gene editing activated T cells is characterized in that the culture medium is RPMI-1640 culture medium, 1000IU/ml of interleukin 7(IL-7) 200-.
2. The culture medium according to claim 1, wherein the culture medium is RPMI-1640 medium, interleukin 7(IL-7)500IU/ml, interleukin 21(IL-21)550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ml and S-2-hydroxyglutaric acid 0.5 mmol/ml.
3. A kit comprising the culture medium of claims 1-2.
4. A method for amplifying gene-editing activated T cells in vitro, comprising the steps of:
(1) pre-culturing the T cells after gene modification, wherein the culture medium is RPMI-1640 culture medium and 80-120ng/ml anti-CD 40 monoclonal antibody;
(2) after pre-culture, adding 1000IU/ml of interleukin 7(IL-7) 200-.
5. The method according to claim 4, wherein the culturing time in the step (1) is 48 hours.
6. The method according to claim 4, wherein in the step (2), the culture medium of the step (1) is added with 500IU/ml of interleukin 7(IL-7), 550IU/ml of interleukin 21(IL-21), 100ng/ml of anti-CD 40 monoclonal antibody, 0.45mmol/ml of serine and 0.5mmol/ml of S-2-hydroxyglutaric acid for efficient amplification culture.
7. The method of claim 4, wherein the genetic modification technique comprises ZEN, TALEN or CRISPR/Cas 9; the genetic modification is carried out by modifying the relevant activating and/or repressing genes in the T-cell.
8. Use of a medium according to any one of claims 1 to 2 for the cultivation of T cells.
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| JP2023503161A (en) | 2019-11-26 | 2023-01-26 | ノバルティス アーゲー | CD19 and CD22 chimeric antigen receptors and uses thereof |
| TW202307210A (en) | 2021-06-01 | 2023-02-16 | 瑞士商諾華公司 | Cd19 and cd22 chimeric antigen receptors and uses thereof |
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| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
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| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
Non-Patent Citations (3)
| Title |
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| Agonistic Antibody to CD40 Boosts the Antitumor Activity of Adoptively Transferred T Cells In Vivo;Chengwen Liu et al.;《J Immunother.》;20120430;第35卷(第3期);第6页倒数第2段 * |
| Serine Is an Essential Metabolite for Effector T Cell Expansion;Eric H. Ma et al.;《Cell Metabolism》;20170207;第25卷;参加摘要 * |
| The immunometabolite S-2-hydroxyglutarate regulates CD8+T-lymphocyte fate;Petros A. Tyrakis et al.;《Nature》;20161208;第540卷;摘要 * |
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