CN106957822A - Cultural method, kit and the application of amplification in vitro gene editing activating T cell - Google Patents

Cultural method, kit and the application of amplification in vitro gene editing activating T cell Download PDF

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CN106957822A
CN106957822A CN201710325993.3A CN201710325993A CN106957822A CN 106957822 A CN106957822 A CN 106957822A CN 201710325993 A CN201710325993 A CN 201710325993A CN 106957822 A CN106957822 A CN 106957822A
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culture medium
amplification
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CN106957822B (en
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邓涛
喻堃
徐国锋
卢铀
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Chengdu Beauty Jessell Biotechnology Co Ltd
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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Abstract

The invention provides a kind of culture medium of amplification in vitro gene editing activating T cell, it is characterised in that the culture medium includes T cell basal medium, also includes：The 1000IU/ml of interleukin-17 (IL 7) 200, the 1000IU/ml of IL-21 (IL 21) 300, the 120ng/ml of anti-CD 40 monoclonal antibody 80, the 1.0mmol/ml of serine 0.2 and the 1.0mmol/ml of 2 hydroxyl glutaric acids of S 0.3.The method of amplification in vitro gene editing activating T cell and the application in terms of T cell is cultivated are carried out present invention also offers the kit containing above-mentioned culture medium and using above-mentioned culture medium., can a large amount of amplification gene editor activating T cell under conditions of it need not add foreign serum and other activating T cell factors using the culture medium of the present invention.The inventive method can activate 500 1000 times of T cell of amplification, hence it is evident that higher than prior art.

Description

Cultural method, kit and the application of amplification in vitro gene editing activating T cell
Technical field
The invention belongs to biotechnology and technical field of cell culture, and in particular to a kind of amplification in vitro gene editing activation Cultural method, kit and its application of T cell.Including but not limited to using genomes such as ZFN, TALEN, CRISPR-CAS9 T cell cultural method, kit and its application of functional activation constructed by editing technique.
Background technology
With deepening continuously for immunological investigation, immunotherapy of tumors obtains huge progress.Immunotherapy of tumors is by swashing Hair or the immune system for transferring patient itself, strengthen tumor microenvironment antineoplastic immune power, so that control and killing tumor cell, Immunotherapy of tumors is also considered as after the fourth-largest oncotherapy technology after operation, radiotherapy, chemotherapy.Nearly ten years, it is thin to adopt Born of the same parents' immunotherapy (adoptive cell therapy, ACT) and immunologic test point therapy (immune checkpoint Therapy breakthrough) is achieved for the immunotherapy of tumors method of representative, good application prospect is shown, indicates The unlatching of oncotherapy New Times.
In immunotherapy of tumors scheme, T lymphocytes are in Central Position.The specific cellular immunity work(of in-vivo tumour Can be lowly that tumour escapes one of major reason for unrestrictedly growing of monitoring of immune system.Cell biological treatment technology will be certainly Body immunocyte is fed back in vivo again by external evoked, differentiation, amplification, bypasses in-vivo tumour immunization barrier mechanism, by swashing The immune response of body is sent out to resist, suppress and kill cancer cell.How to obtain sufficient amount in vitro is available for the T of clinical practice thin Born of the same parents are primary problems faceds in tumour adoptive immunotherapy.
Genome editor (Genome editing) technology is to carry out fixed point transformation to DNA sequence dna in genomic level to repair The Genetic Manipulative Technology of decorations, the technology is described as " booster of genome times afterwards comprehensively life science ".Genome editor's skill The principle of art is, by building an artificial incision enzyme, DNA to be cut off in target site, produces DNA double chain fracture (Double- Strand break, DSB), and then the DNA repair systems progress non-homologous end joining (Nonhomologous in inducing cell End joining, NHEJ) and homologous recombination repair (Homologous recombination, HR).Pass through both reparation ways Footpath, genome editing technique can realize that fixed point gene knockout, specific mutagenesis are introduced and pointed decoration.But in genome editor's skill Before art is undiscovered, scientists can only be oriented modification by way of homologous recombination to genome.But this side Formula depends on nature restructuring unduly, extremely inefficient, it is impossible to meet the growing scientific research demand of the mankind.In the case, gene is compiled The technology of collecting is arisen at the historic moment.In in recent years, artificial endonucleases (engineered endonuclease, EEN) technology it is emerging Rise and make it that gene editing becomes simple possible, now conventional EEN technologies are mainly Zinc finger nuclease (zinc finger Nuclease, ZFN), class activating transcription factor effector nuclease (transcription activator-like Effector nuclease, TALEN) and short palindrome repeat techniques (the Clustered regulatory in Regularity interval Interspaced short palindromic repeat, CRISPR).
At present, in tumour cell immunization therapy, T cell is the immune core roles of antitumor cell, however, tumour is thin Born of the same parents are often expressed after some parts are combined with T cell, are suppressed activation, propagation and the killing to tumour of T cell, are sent out its function It is raw disorderly and exhausted, so as to reach the purpose of immune evasion.By gene editing technology by the specific gene or base in T cell Because group knocks out, so as to preferably play the purpose of killing tumor cell.
The content of the invention
It is an object of the invention to improve the amplification rate of the T cell by gene editing technical finesse, reduce amplification Time, provide it is a kind of being capable of economical, the simply and efficiently cultural method of amplification gene editor activating T cell, culture medium and its should With so as to improve the therapeutic action of the gene editing activating T cell to tumour or Other diseases.
A kind of culture medium for amplification in vitro gene editing activating T cell that the present invention is provided, is cultivated comprising T cell basis Base, is also included：It is interleukin-17 (IL-7) 200-1000IU/ml, IL-21 (IL-21) 300-1000IU/ml, anti- CD40 monoclonal antibodies 80-120ng/ml, serine 0.2-1.0mmol/ml and S-2- hydroxyl glutaric acid 0.3-1.0mmol/ml.
Preferably, the culture medium includes T cell basal medium, also includes：Interleukin-17 (IL-7) 500IU/ml, IL-21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ Ml and S-2- hydroxyl glutaric acids 0.5mmol/ml.
Preferably, the basal medium is RPMI-1640 culture mediums.
The above-mentioned culture medium of the present invention can be not only used for culturing gene editor's activating T cell, can be used for culture common T cell.
Therefore it is also an object of the present invention to provide the application using the culture medium in terms of T cell is cultivated.
A kind of method for amplification in vitro gene editing activating T cell that the present invention is provided, comprises the following steps：
(1) preculture is carried out to the T cell after genetic modification, during culture, used medium includes basal medium, also Include anti-CD 40 monoclonal antibody 80-120ng/ml；
(2) after preculture, interleukin-17 (IL-7) 200-1000IU/ml is added into step (1) culture medium, it is white thin Born of the same parents' interleukin 21 (IL-21) 300-1000IU/ml, anti-CD 40 monoclonal antibody 80-120ng/ml, serine 0.2-1.0mmol/ml With S-2- hydroxyl glutaric acid 0.3-1.0mmol/ml, efficient amplification culture is carried out.
Preferably, in step (1), basal medium is RPMI-1640 culture mediums.
Preferably, in step (1), incubation time is 48 hours.
Preferably, in step (2), interleukin-17 (IL-7) 500IU/ is added into step (1) culture medium Ml, IL-21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ml and S- 2- hydroxyl glutaric acid 0.5mmol/ml, carry out efficient amplification culture.
Optionally, the technology of the genetic modification includes ZEN, TALE N or CRISPR/Cas9.
It is the associated activation and/or suppressor modified in T cell when carrying out the genetic modification.
CD40 is one of T cell surface membrane protein receptors, CD40 and its ligand binding can by activating mTORC1 paths, Play a part of PD-1 antagonists, thus play a part of promote T cell fast breeding, the present invention using anti-CD 40 antibodies with CD40 combines to activate mTORC1 paths, promotes T cell fast breeding.
The serine and S-2- hydroxyls glutaric acid added in culture medium of the present invention is all that T cell is carried out during fast breeding Material necessary to energetic supersession, T cell can be greatly improved by additionally adding a certain amount of serine and S-2- hydroxyls glutaric acid Growth rate.
, can be with conditions of it need not add foreign serum and other activating T cell factors using the culture medium of the present invention A large amount of amplification gene editor's activating T cells, the abductive approach of the culture medium rapidly and efficiently, economic simple, effect stability.In addition, Shown according to the flow cytomery result of industry universal, the method for the amplification in vitro gene editing activating T cell can Activation 500-1000 times of T cell of amplification, hence it is evident that higher than prior art.
Embodiment
Following embodiments are further illustrating for present invention, but the present invention is not limited to these specific embodiment parties Formula.Gene editing technology used mainly includes ZFN, TALEN, CRISPR- in gene editing activating T cell of the present invention Cas9 etc., but the invention is not restricted to use three of the above gene editing technology.A kind of amplification in vitro gene editing of the present invention Cultural method, kit and its application of activating T cell are mainly used in T cell in-vitro separation, induction, differentiation, culture, amplification Deng；Its biological products handled includes human peripheral, Cord blood, hydrothorax, ascites etc.；Clinical cytology biology is can apply to control Treat, including lung cancer, kidney, melanoma, leukaemia, breast cancer, the carcinoma of the rectum, stomach cancer, cancer of the esophagus, cervical carcinoma, oophoroma, marrow The malignancy diseases such as cancer, malignant lymphoma, can also grind applied to basic medical research, clinical application research and biotechnology Study carefully, it is particularly possible to the research acted on applied to immune system research and tumor-killing.Specific embodiment is as follows：
Embodiment 1 (experimental group)
The separation of people PD-1 transgenic T cells and efficient amplification culture are knocked out with CRISPR-Cas9 technologies
Now using human peripheral as processing sample, people's PD-1 genes are knocked out with CRISPR-Cas9 technologies, PD-1 genes are built T cell is knocked out, and external efficient amplification culture is carried out to the gene editing activating T cell.Comprise the following steps that：
Step one：PMNC is isolated from blood
Donor 80~100ml of venous blood is directly extracted, anti-coagulants -- heparin is added；
By the blood sample of collection, 2000rpm centrifuges 10min at room temperature, standby during careful absorption upper plasma layer culture；Blood All is restored to original volume, mixes；Diluted blood is slowly added on Ficoll, 1000rpm centrifugations 20min；Draw separating liquid circle Face milky mononuclearcell layer, centrifuge washing 2 times obtains PBMC.
Step 2：T cell is separated from PBMC
Taking the PBMC of separation, add basal medium RPMI-1640 and be made into cell suspension, adjustment cell concentration is 1~2 × 106Individual/ml, 37 DEG C, 5%CO2It is T cell that suspension cell is collected after being incubated 2 hours.
Step 3：People's PD-1 genes in T cell are knocked out with CRISPR-Cas9 technologies
Build and carry PD-1 gene sgRNA oligonucleotide viral vectors
According to sgRNA design principle, target sequences of the design sgRNA on PD-1 genes synthesizes sgRNA oligonucleotides Double-strand, mixing is connected with the slow virus carrier of linearisation after 95 DEG C of annealing 5min, obtains and carry PD-1 genes sgRNA afterwards Oligonucleotide viral vectors；
Build and carry Cas9- nuclease relevant carriers
Cas9 nucleic acid enzyme clones are entered in relevant carriers, Cas9- nucleic acid zymophores are obtained；
Transduction T cell
The T cell for being collected the synchronous Transduction protocol two of corresponding sgRNA and Cas9- nucleases using ad hoc fashion, is completed People's PD-1 genes are knocked out, that is, obtains and knocks out PD-1 transgenic T cells.
Step 4：Preculture PD-1 gene knockout T cells
PD-1 gene knockout T cells are collected, suspension is incubated in basal medium RPMI-1640, adds anti-CD 40 antibodies 80~120ng/ml, 37 DEG C, 5%CO2It is incubated culture 48h.
Step 5：The efficient amplification culture of PD-1 gene knockout T cells
Interleukin-17 (IL-7) 500IU/ml, leucocyte are added into the T cell nutrient solution after step 4 culture 48h Interleukin 21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ml and S-2- hydroxyl penta 2 Sour 0.5mmol/ml continues to cultivate.Measured every three days with nutrient solution half and change liquid, half amount changes liquid to take the celliferous training of half Base is supported, is collected by centrifugation after cell and removes culture medium, adds and contains interleukin-17 (IL-7) 500IU/ml, IL-21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ml and S-2- hydroxyl glutaric acid 0.5mmol/ml RPMI-1640 culture mediums adjustment cell concentration is 1 × 106Individual/ml.
Since step 5, after culture 48h, T cell is transferred in T175 blake bottles and continues to cultivate.To ensure that cell has Sufficient growing space.
Since step 5, after cultivating 14~21 days, T cell is collected, after T cell is centrifuged, brine is used Three times, that is, the T cell activated.
Comparative example 1：It is not added with anti-CD 40 monoclonal antibody, serine, S-2- hydroxyls glutaric acid (control group 1)
Except in step 4 in embodiment 1 and step 5 without anti-CD 40 monoclonal antibody, serine, S-2- hydroxyls penta 2 Beyond acid, other concrete operation steps such as embodiment 1.
Comparative example 2 (control group 2)：
Except the concentration of the anti-CD 40 monoclonal antibody added in step 4 in embodiment 1 and step 5 is 70ng/ml, silk The concentration of propylhomoserin is other concrete operation steps such as embodiment 1 beyond 1.1mmol/ml.
Comparative example 3 (control group 3)
Except the concentration of anti-CD 40 monoclonal antibody added in step 4 in embodiment 1 and step 5 be 130ng/ml, The concentration of S-2- hydroxyl glutaric acids is other concrete operation steps such as embodiment 1 beyond 0.25mmol/ml.Experiment to embodiment 1 Group and the gene editing T cell of the gained of control group 1~3 carry out amplification times detection.
The 1st, take 200 μ l cells within 7,14,21 days, blow and beat into individual cells, utilize Counterstar automatic blood cell meters Number instrument is counted, the TCS before the TCS/1st day culture that cells expanded=same day counts.Experimental result Such as table 1.
Change (× 10 of the T cell of table 1 in different time cell quantity8/L)

Claims (10)

1. a kind of culture medium of amplification in vitro gene editing activating T cell, it is characterised in that the culture medium includes T cell base Basal culture medium, is also included：Interleukin-17 (IL-7) 200-1000IU/ml, IL-21 (IL-21) 300-1000IU/ Ml, anti-CD 40 monoclonal antibody 80-120ng/ml, serine 0.2-1.0mmol/ml and S-2- hydroxyl glutaric acid 0.3-1.0mmol/ ml。
2. culture medium according to claim 1, it is characterised in that the culture medium includes T cell basal medium, also wraps Contain：Interleukin-17 (IL-7) 500IU/ml, IL-21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, serine 0.45mmol/ml and S-2- hydroxyl glutaric acid 0.5mmol/ml.
3. culture medium according to claim 1 or 2, it is characterised in that the basal medium is cultivated for RPMI-1640 Base.
4. include the kit of the culture medium described in claims 1 to 3.
5. a kind of method of amplification in vitro gene editing activating T cell, it is characterised in that methods described comprises the following steps：
(1) preculture is carried out to the T cell after genetic modification, during culture, used medium includes basal medium, also includes Anti-CD 40 monoclonal antibody 80-120ng/ml；
(2) after preculture, interleukin-17 (IL-7) 200-1000IU/ml, interleukin 8 are added into step (1) culture medium 21 (IL-21) 300-1000IU/ml of element, anti-CD 40 monoclonal antibody 80-120ng/ml, serine 0.2-1.0mmol/ml and S- 2- hydroxyl glutaric acid 0.3-1.0mmol/ml, carry out efficient amplification culture.
6. method according to claim 5, it is characterised in that in step (1), basal medium is cultivated for RPMI-1640 Base.
7. method according to claim 5, it is characterised in that in step (1), incubation time is 48 hours.
8. method according to claim 5, it is characterised in that in step (2), is added white thin into step (1) culture medium Born of the same parents' interleukin 7 (IL-7) 500IU/ml, IL-21 (IL-21) 550IU/ml, anti-CD 40 monoclonal antibody 100ng/ml, silk Propylhomoserin 0.45mmol/ml and S-2- hydroxyl glutaric acid 0.5mmol/ml, carries out efficient amplification culture.
9. method according to claim 5, it is characterised in that the technology of the genetic modification includes ZEN, TALE N or CRISPR/Cas9；It is the associated activation and/or suppressor modified in T cell when carrying out the genetic modification.
10. application of any one of claims 1 to 3 culture medium in terms of T cell is cultivated.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475292A (en) * 2017-08-02 2017-12-15 山东百福基因科技有限公司 The preparation method of the gene defection type T lymphocyte preparations of PD 1
WO2021108613A1 (en) 2019-11-26 2021-06-03 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
WO2022254337A1 (en) 2021-06-01 2022-12-08 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543170A (en) * 2015-12-31 2016-05-04 中山大学 Composition capable of stimulating expansion of T cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543170A (en) * 2015-12-31 2016-05-04 中山大学 Composition capable of stimulating expansion of T cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENGWEN LIU ET AL.: "Agonistic Antibody to CD40 Boosts the Antitumor Activity of Adoptively Transferred T Cells In Vivo", 《J IMMUNOTHER.》 *
ERIC H. MA ET AL.: "Serine Is an Essential Metabolite for Effector T Cell Expansion", 《CELL METABOLISM》 *
PETROS A. TYRAKIS ET AL.: "The immunometabolite S-2-hydroxyglutarate regulates CD8+T-lymphocyte fate", 《NATURE》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475292A (en) * 2017-08-02 2017-12-15 山东百福基因科技有限公司 The preparation method of the gene defection type T lymphocyte preparations of PD 1
WO2021108613A1 (en) 2019-11-26 2021-06-03 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
WO2022254337A1 (en) 2021-06-01 2022-12-08 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof

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