CN112063653A - Method for preparing NK (natural killer) sample cells based on electrotransfer reprogramming plasmid - Google Patents
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Abstract
The invention provides a method for preparing NK-like cells based on electrotransfer reprogramming plasmids, which comprises the following steps: (1) activating T cells with a combination of anti-human CD3 antibody and anti-human CD28 antibody; (2) and (3) electrically transferring the CRISPR/Cas9 plasmid targeting the Bcl11b gene into the activated T cell to obtain the NK-like cell. The invention adopts an electrotransformation method to introduce CRISPR/Cas9 plasmid of the targeted Bcl11b gene into activated T cells, and reasonably sets electrotransformation conditions, so that a gene editing system is transferred into primary T cells to carry out efficient gene editing, the cytotoxicity is obviously reduced, and the NK-like cells with killing effect are prepared by matching with reasonable culture amplification conditions.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for preparing NK-like cells based on electrotransformation reprogramming plasmids.
Background
In recent years, cellular immunotherapy has shown great potential in cancer treatment, and is considered to be the most promising method for overcoming cancer, and among them, T cell-based immunotherapy technology has received extensive attention and is the current research hotspot. To overcome the limitations of the Major Histocompatibility Complex (MHC), researchers have studied the mechanism of activating differentiation of T cells, attempting to genetically modify them to obtain T cells with enhanced antitumor activity.
Li et al have successfully reprogrammed mouse T cells into NK-like cells (ITNK cells) (Li, P.et al. replication of T cells to natural killers-like cells Bcl11b deletion. science 329,85-89 (2010)), and recently successfully reprogrammed human T cells into ITNK cells using CRISPR/Cas9 technology.
The CRISPR/Cas9 is a gene editing tool which is most deeply researched and mature in application, and has been successfully applied to gene editing of various cell lines. However, the editing efficiency of the method for primary cells is very low, and the method for gene editing by transferring Cas9 and gRNA into primary T cells has the problems of low targeting efficiency and high cytotoxicity, and the application of the CRISPR technology to primary cells is severely limited.
Therefore, there is a need to improve the process of transferring the CRISPR/Cas9 system into primary T cells, not only reduce the toxicity of the gene editing system to the primary T cells, but also improve the reprogramming efficiency and enhance the tumor killing function of the reprogrammed ITNK cells.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a method for preparing NK-like cells based on electrotransformation reprogramming plasmids, the method electrically transfers CRISPR/Cas9 plasmids with the function of knocking out Bcl11b genes into activated T cells, and cultures the transformed cells by adopting the combination of cytokines IL-12, IL-15 and IL-18, so that the gene editing efficiency is high, and the NK-like cells which effectively recognize and kill tumor cells with high purity are obtained.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing NK-like cells based on an electrotransformation reprogramming plasmid, said method comprising the steps of:
(1) activating T cells with a combination of anti-human CD3 antibody and anti-human CD28 antibody;
(2) and (3) electrically transferring the CRISPR/Cas9 plasmid targeting the Bcl11b gene into the activated T cell to obtain the NK-like cell.
According to the invention, firstly, the anti-human CD3 antibody and the anti-human CD28 antibody are adopted to activate the T cell, then the CRISPR/Cas9 plasmid targeting the Bcl11b gene is electrically transferred into the activated T cell, and all conditions are cooperated, so that the high-efficiency transfer of the gene editing system into the primary T cell is ensured, the gene editing of the T cell based on the gene editing system is realized, and the preparation of the high-purity NK-like cell for identifying and killing the tumor cell is facilitated.
Preferably, the T cells are derived from peripheral blood and/or umbilical cord blood.
Preferably, the molar ratio of the anti-human CD3 antibody to the anti-human CD28 antibody is (1-5): 1, and may be, for example, 1:1, 2:1, 3:1, 4:1 or 5: 1.
In the invention, the combination of the anti-human CD3 antibody and the anti-human CD28 antibody with the molar ratio of (1-5): 1 has the best activation effect on the T cells, is beneficial to promoting the CRISPR/Cas9 plasmid to be introduced into the T cells and reducing the toxicity of the electrotransformation operation on the T cells, and the combination of the anti-human CD3 antibody and the anti-human CD28 antibody with the ratio of less than or greater than (1-5): 1 cannot realize sufficient activation on the T cells.
Preferably, after the T cell is activated for 3-6 days, for example, 3 days, 4 days, 5 days or 6 days, the CRISPR/Cas9 plasmid targeting the Bcl11b gene is electrotransferred into the T cell, and after 3-6 days of T cell activation, the activated T cell is in a rapid proliferation state, and the electrotransfer of the plasmid is performed at this time point, which is favorable for improving the efficiency of electrotransferred into the T cell of the CRISPR/Cas9 plasmid targeting the Bcl11b gene.
Preferably, the CRISPR/Cas9 plasmid targeting the Bcl11b gene contains a sgRNA expression cassette and a Cas9 protein expression cassette, and the sgRNA expression cassette and the Cas9 protein expression cassette are inserted after the T7 promoter of the PX458-gBCL11b vector to construct a plasmid for knocking out the Bcl11b gene.
Preferably, the sgRNA comprises the nucleic acid sequence shown in SEQ ID NO. 1;
SEQ ID NO:1：gaccatgaactgctcacttg。
preferably, the voltage of the electric transfer is 800-2500V, such as 800V, 1000V, 1100V, 1200V, 1300V, 1400V, 1500V, 1600V, 1700V, 1800V, 1900V, 2000V, 2100V, 2200V, 2300V, 2400V or 2500V.
Preferably, the time of the electrical transfer is 1-8 ms, for example, 1ms, 2ms, 3ms, 4ms, 5ms, 6ms, 7ms or 8 ms.
Preferably, for a 2mm electric shock cup, the voltage of the electric shock cup is 1000-2000V, and the time is 1-4 ms.
According to the invention, the voltage of electrotransformation is defined to be 800-2500V, the time of electrotransformation is defined to be 1-8 ms, and further preferably for a 2mm electric shock cup, the voltage of electrotransformation is 1000-2000V, and the time is 1-4 ms, the CRISPR/Cas9 plasmid is electrotransferred into T cells activated for 3-6 days by an anti-human CD3 antibody and an anti-human CD28 antibody under the electrotransferred condition, and the conditions are matched with each other, so that the efficiency of electrotransferred CRISPR/Cas9 plasmid into T cells is obviously improved, the cytotoxicity of the electrotransferred CRISPR/Cas9 plasmid to the T cells is reduced, and the preparation of NK-like cells with high purity and obvious tumor killing activity is facilitated.
Preferably, the step (2) is followed by a step of culturing the NK-like cells using a medium.
Preferably, the medium is supplemented with a cytokine composition.
Preferably, the cytokine composition includes IL-12, IL-15 and IL-18.
Preferably, the concentration of IL-12 in the culture medium is 1-100 ng/mL, for example, 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.
Preferably, the concentration of IL-15 in the culture medium is 1-100 ng/mL, for example, 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.
Preferably, the concentration of IL-18 in the culture medium is 1-100 ng/mL, for example, 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.
Preferably, the cytokine composition is added at the beginning of culturing the NK-like cells using the medium and is removed at 24-72 hours after the initiation of culturing.
According to the invention, the NK-like cells are cultured for 24-72 hours by using the culture medium added with 1-100 ng/mL IL-12, 1-100 ng/mL IL-15 and 1-100 ng/mL IL-18, so that a large amount of high-purity NK-like cells can be rapidly obtained, and cytokine compositions IL-12, IL-15 and IL-18 in the culture medium are removed after 24-72 hours, so that over-activation of the NK-like cells is avoided, and the NK-like cells obtained under the conditions are high in yield, purity and activity.
As a preferred technical scheme, the invention provides a method for preparing NK-like cells based on electrotransfer reprogramming plasmids, which comprises the following steps:
(1) separating mononuclear cells from peripheral blood and/or umbilical cord blood and sorting the mononuclear cells to obtain T cells, and activating the T cells by adopting the combination of an anti-human CD3 antibody and an anti-human CD28 antibody in a molar ratio of (1-5): 1;
(2) after the T cell is activated for 3-6 days, the CRISPR/Cas9 plasmid of a targeted Bcl11b gene containing a sgRNA expression frame and a Cas9 protein expression frame is electrically transferred into the activated T cell, wherein the sgRNA comprises a nucleic acid sequence shown as SEQ ID NO. 1, the voltage of the electric transfer is 1000-2000V in a 2mm electric shock cup, and the time is 1-4 ms;
(3) and (3) culturing the cells after the electrotransformation for 24-72 hours by adopting a culture medium added with 1-100 ng/mL IL-12, 1-100 ng/mL IL-15 and 1-100 ng/mL IL-18, removing the IL-12, IL-15 and IL-18 in the culture medium, and continuously culturing and amplifying to obtain the NK-like cells.
In a second aspect, the present invention provides an NK-like cell prepared by the method of the first aspect.
In a third aspect, the invention provides the use of the NK-like cells of the second aspect in the preparation of a medicament for the immunotherapy of tumors.
Preferably, the tumor comprises a solid tumor and/or a hematological tumor.
Preferably, the solid tumor includes any one of or a combination of at least two of liver cancer, lung cancer, breast cancer, stomach cancer, nephroblastoma, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor of pancreas, retinoblastoma, pancreatic cancer, uterine fibroids, cervical cancer, or thyroid cancer.
Preferably, the hematological tumor comprises any one of acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, acute lymphoid leukemia, non-hodgkin lymphoma, plasmablast lymphoma or plasmacytoid dendritic cell tumor or a combination of at least two thereof.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, firstly, an anti-human CD3 antibody and an anti-human CD28 antibody with a molar ratio of (1-5): 1 are adopted to activate T cells, CRISPR/Cas9 plasmids of targeted Bcl11b genes are electrically transferred into the activated T cells after 3-6 days, and all conditions are cooperated to ensure that a gene editing system is efficiently transferred into primary T cells, so that the gene editing of the T cells based on the gene editing system is realized;
(2) according to the invention, the voltage of electric transfer is set to be 1000-2000V according to the activation state of the T cell, and the time is 1-4 ms, so that the efficiency of electric transfer of CRISPR/Cas9 plasmid into the T cell is obviously improved, and the cytotoxicity of the electric transfer CRISPR/Cas9 plasmid on the T cell is reduced;
(3) according to the invention, the NK-like cells are cultured for 24-72 hours by using the culture medium added with 1-100 ng/mL IL-12, 1-100 ng/mL IL-15 and 1-100 ng/mL IL-18, so that a large amount of high-purity NK-like cells can be rapidly obtained, and meanwhile, the NK-like cells are prevented from being excessively activated;
(4) the high-purity NK-like cells for identifying and killing tumor cells are prepared, have obvious in-vitro killing effect, secrete a large amount of IFN-gamma cytokines after being co-cultured with target cells, and play a role in killing the cells.
Drawings
FIG. 1 shows NK cell ratios of NK cells prepared in examples 2 to 8 and comparative examples 1 to 2;
FIG. 2 is a graph of in vitro killing of different NK-like cells;
FIG. 3 shows the secretion of IFN-. gamma.of different NK-like cells co-cultured with human myeloid leukemia cell line KG 1.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 vector construction
In order to construct a CRISPR/Cas9 plasmid for targeting and knocking out a Bcl11b gene, sgRNA (SEQ ID NO:1) is firstly designed according to the Bcl11b gene, and restriction enzyme cutting sites EcoRI and SalI are added at the 5 'end and the 3' end of a sequence after artificial synthesis;
the synthesized sequence fragment is cut by EcoRI and SalI, then is connected to a PX458-gBCL11b vector containing a T7 promoter, and the sequencing verifies that the recombinant plasmid is successfully constructed.
Example 2 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 100ng/mL anti-human CD3 antibody and 100ng/mL anti-human CD28 antibody;
(2) after 3 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding CRISPR/Cas9 plasmid with the concentration of 40 mu M, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 800V, and the time is 4 ms;
(3) after the electroporation, the cells were cultured for 72 hours in a complete medium T551-H3 containing 1ng/mL IL-12, 1ng/mL IL-15 and 1ng/mL IL-18, the cells were centrifuged at a low speed, and the culture was continued for 12 hours by replacing the complete medium with fresh T551-H3 to obtain NK-like cells.
Example 3 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL of T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 300ng/mL of anti-human CD3 antibody and 100ng/mL of anti-human CD28 antibody;
(2) after 3 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding 80 mu M CRISPR/Cas9 plasmid, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 1200V, and the time is 3 ms;
(3) after the electroporation, the cells were cultured for 36 hours in a complete medium T551-H3 containing 50ng/mL IL-12, 50ng/mL IL-15 and 50ng/mL IL-18, the cells were centrifuged at a low speed, and the cells were further cultured for 48 hours by replacing the complete medium with fresh T551-H3 to obtain NK-like cells.
Example 4 preparation of NK-like cells
(1) Will be 1 × 107The individual T cells were resuspended in 3mL of T cell culture medium, seeded into one well of a 6-well plate, and a combination of 400ng/mL anti-human CD3 antibody and 100ng/mL anti-human CD28 antibody was addedActivating;
(2) after 5 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding CRISPR/Cas9 plasmid with the concentration of 120 mu M, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 2000V, and the time is 1 ms;
(3) after electrotransformation, the cells were cultured for 24 hours in a complete medium T551-H3 containing 100ng/mL IL-12, 100ng/mL IL-15 and 100ng/mL IL-18, the cells were centrifuged at a low speed, and the cells were further cultured for 60 hours by replacing the complete medium with fresh T551-H3 to obtain NK-like cells.
Example 5 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL of T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 500ng/mL anti-human CD3 antibody and 100ng/mL anti-human CD28 antibody;
(2) after 6 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding 80 mu M CRISPR/Cas9 plasmid, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 2000V, and the time is 2 ms;
(3) and culturing the cells after electrotransformation for 72H by adopting a T551-H3 complete medium containing 10ng/mL IL-12 and 50ng/mL IL-15, centrifuging the cells at a low speed, and replacing a fresh T551-H3 complete medium to continuously culture for 12H to obtain NK-like cells.
Example 6 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL of T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 200ng/mL anti-human CD3 antibody and 100ng/mL anti-human CD28 antibody;
(2) counting activated cells, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding CRISPR/Cas9 plasmid with the concentration of 40 mu M, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-nucleofectTM X Unit (single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 1000V, and the time is 4 ms;
(3) after the electroporation, the cells were cultured for 72 hours in a complete medium T551-H3 containing 10ng/mL IL-12, 10ng/mL IL-15 and 10ng/mL IL-18, the cells were centrifuged at a low speed, and the culture was continued for 12 hours by replacing the complete medium with fresh T551-H3 to obtain NK-like cells.
Example 7 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL of T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 300ng/mL of anti-human CD3 antibody and 100ng/mL of anti-human CD28 antibody;
(2) after 3 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding 80 mu M CRISPR/Cas9 plasmid, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 1200V, and the time is 3 ms;
(3) the cells after the electroporation were cultured for 84 hours in a fresh complete medium of T551-H3 to obtain NK-like cells.
Example 8 preparation of NK-like cells
(1) Will be 1 × 107Resuspending individual T cells in 3mL of T cell culture medium, plating one well of a 6-well plate, and activating by adding a combination of 400ng/mL of anti-human CD3 antibody and 100ng/mL of anti-human CD28 antibody;
(2) after 5 days, taking out suspended cells for counting, centrifuging for 10min at 300 Xg, resuspending the cell sediment by using 10mL of Opti-MEM, centrifuging for 10min at 300 Xg, resuspending the cell sediment in 100 mu L of Opti-MEM, adding CRISPR/Cas9 plasmid with the concentration of 120 mu M, uniformly mixing, transferring to an electric Lonza electric shock cup, placing the electric shock cup in a Lonza 4D-Nuclear electric shock TM X Unit (in a single electric shock cup module), and carrying out electric transfer, wherein the voltage of the electric transfer is set to be 2000V, and the time is 1 ms;
(3) the transfected cells were cultured for 84H in complete medium T551-H3 containing 100ng/mL IL-12, 100ng/mL IL-15 and 100ng/mL IL-18 to give NK-like cells.
Comparative example 1
Compared with example 2, the T cell of comparative example 1 is directly transformed into CRISPR/Cas9 plasmid without activation of anti-human CD3 antibody and anti-human CD28 antibody, and other conditions are the same as example 2.
Comparative example 2
In contrast to example 2, comparative example 2 did not electroporate the CRISPR/Cas9 plasmid into activated T cells as a blank, with the other conditions being the same as in example 2.
Proportion of NK-like cells
The ratio of INTK cells (expressing both the T cell marker CD3 and the NK cell markers NKp46 and NKp30) prepared in the different examples and comparative examples was examined by flow cytometry.
As shown in FIG. 1, the ratio of NK-like cells prepared in examples 2 to 8 was significantly higher than that of comparative examples 1 to 2, and comparative example 1 did not activate T cells, and thus the electroporation efficiency was low and the ratio of NK-like cells was low.
In different examples, the cell survival rates and the NK-like cell ratios of examples 2-5 and 8 are higher than those of examples 6-7, because the activated T cells are directly transformed by electricity in example 6, the number of the activated T cells is low, the transformation efficiency is affected, and the transformed cells are not cultured in the medium containing the cytokine composition in example 7, so that the cells cannot recover from the transformed cells in time, and a certain degree of cell death is caused.
Killing of NK-like cells and the ability to secrete IFN γ
The NK-like cells of examples 2 to 8 and comparative examples 1 to 2 were mixed with 5X 10 cells, respectively3Co-culturing a human myeloid leukemia cell line KG1 in a U-shaped 96-well plate, wherein the ratio (E: T) of effector cells to target cells is 4:1, and each group of experiments is repeated for 3 times;
after 18 hours of co-culture, 100. mu.L/well Luciferase substrate (1X) was added to a 96-well plate, the cells were resuspended and mixed, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of NK samples of different examples and comparative examples on KG1 was compared in vitro by the Luciferase (Luciferase) quantitative killing efficiency evaluation method, and the killing ratio calculation formula was as follows:
100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)
As shown in FIG. 2, the in vitro killing efficiency of NK-like cells prepared in example was higher than that of NK-like cells prepared in comparative example, wherein the in vitro killing efficiency of NK-like cells prepared in example 3 and example 4 was the highest, and it is noted that example 8, although high purity NK-like cells were obtained, the killing performance was decreased due to excessive activation by IL-12, IL-15 and IL-18; comparative examples 1-2 influence the killing efficiency of NK-like cells due to unreasonable conditions.
And detecting the co-culture supernatant of the effector cells and the target cells by adopting an IFN-gamma ELISA detection kit.
As shown in FIG. 3, the results of co-culturing the NK-like cells of the example with KG1 revealed that IFN-. gamma.was secreted in a larger amount than the comparative example, and the results were consistent with the killing effect.
In conclusion, the CRISPR/Cas9 plasmid targeting the Bcl11b gene is introduced into the activated T cell by adopting an electrical transformation method, and electrical transformation conditions are reasonably set, so that a gene editing system is transferred into the primary T cell to carry out efficient gene editing, the cytotoxicity is obviously reduced, and the NK-like cell with the killing effect is prepared by matching with reasonable culture amplification conditions.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Guangdong Shoutai biomedical science and technology Co., Ltd
<120> method for preparing NK-like cells based on electrotransformation reprogramming plasmid
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
Claims (10)
1. A method for preparing NK-like cells based on an electroporation reprogramming plasmid, comprising the steps of:
(1) activating T cells with a combination of anti-human CD3 antibody and anti-human CD28 antibody;
(2) and (3) electrically transferring the CRISPR/Cas9 plasmid targeting the Bcl11b gene into the activated T cell to obtain the NK-like cell.
2. The method of claim 1, wherein the T cells are derived from peripheral blood and/or umbilical cord blood.
3. The method of claim 1 or 2, wherein the molar ratio of the anti-human CD3 antibody to the anti-human CD28 antibody is (1-5): 1.
4. The method according to any one of claims 1 to 3, wherein the CRISPR/Cas9 plasmid targeting the Bcl11b gene is electroporated into the T cell 3 to 6 days after the T cell is activated.
5. The method of any one of claims 1-4, wherein the CRISPR/Cas9 plasmid targeting the Bcl11b gene contains a sgRNA expression cassette and a Cas9 protein expression cassette;
preferably, the sgRNA comprises the nucleic acid sequence shown in SEQ ID NO. 1.
6. The method according to any one of claims 1 to 5, wherein the voltage of the electrical transfer is 800 to 2500V;
preferably, the electric conversion time is 1-8 ms;
preferably, for a 2mm electric shock cup, the voltage of the electric shock cup is 1000-2000V, and the time is 1-4 ms.
7. The method according to any one of claims 1 to 6, further comprising a step of culturing the NK-like cells with a culture medium after step (2);
preferably, a cytokine composition is added to the medium;
preferably, the cytokine composition includes IL-12, IL-15 and IL-18;
preferably, the concentration of the IL-12 in the culture medium is 1-100 ng/mL;
preferably, the concentration of the IL-15 in the culture medium is 1-100 ng/mL;
preferably, the concentration of the IL-18 in the culture medium is 1-100 ng/mL;
preferably, the cytokine composition is added at the beginning of culturing the NK-like cells using the medium and is removed at 24-72 hours after the initiation of culturing.
8. The method according to any one of claims 1-7, characterized in that the method comprises the steps of:
(1) separating mononuclear cells from peripheral blood and/or umbilical cord blood and sorting the mononuclear cells to obtain T cells, and activating the T cells by adopting the combination of an anti-human CD3 antibody and an anti-human CD28 antibody in a molar ratio of (1-5): 1;
(2) after the T cell is activated for 3-6 days, the CRISPR/Cas9 plasmid of a targeted Bcl11b gene containing a sgRNA expression frame and a Cas9 protein expression frame is electrically transferred into the activated T cell, wherein the sgRNA comprises a nucleic acid sequence shown as SEQ ID NO. 1, the voltage of the electric transfer is 1000-2000V in a 2mm electric shock cup, and the time is 1-4 ms;
(3) and (3) culturing the cells after the electric transformation for 24-72 hours by adopting a culture medium added with 1-100 ng/mL IL-12, 1-100 ng/mL IL-15 and 1-100 ng/mLIL-18, removing the IL-12, IL-15 and IL-18 in the culture medium, and continuously culturing and amplifying to obtain the NK-like cells.
9. An NK-like cell produced by the method of any one of claims 1 to 8.
10. Use of the NK-like cells of claim 9 for the preparation of a medicament for the immunotherapy of tumors;
preferably, the tumor comprises a solid tumor and/or a hematological tumor;
preferably, the solid tumor includes any one or a combination of at least two of liver cancer, lung cancer, breast cancer, stomach cancer, nephroblastoma, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor of pancreas, retinoblastoma, pancreatic cancer, uterine fibroids, cervical cancer, or thyroid cancer;
preferably, the hematological tumor comprises any one of acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, acute lymphoid leukemia, non-hodgkin lymphoma, plasmablast lymphoma or plasmacytoid dendritic cell tumor or a combination of at least two thereof.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574952A (en) * | 2019-09-30 | 2021-03-30 | 英基生物医药(香港)有限公司 | Engineered human immune cell, preparation method and application thereof |
| WO2023038475A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Method for producing directly reprogrammed natural killer cells and use thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120282646A1 (en) * | 2009-10-26 | 2012-11-08 | Korea Research Institute Of Bioscience And Biotechnology | Method for activating a natural killer cell by adjusting the expression of the socs2 gene |
| CN104928242A (en) * | 2015-05-21 | 2015-09-23 | 武汉汉密顿生物科技股份有限公司 | Culturing method of NK (natural killer) cell |
| CN108220235A (en) * | 2016-12-22 | 2018-06-29 | 细胞邦(北京)生物技术有限公司 | A kind of Activated in Vitro expands people's natural kill（NK）The method of cell and its special cultivating system |
| CN109161527A (en) * | 2018-09-25 | 2019-01-08 | 深圳市五零生命科技有限公司 | A kind of efficient NK methods for cell expansion |
| CN109810944A (en) * | 2019-01-24 | 2019-05-28 | 安徽瑞达健康产业有限公司 | A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell |
| CN111607569A (en) * | 2020-06-01 | 2020-09-01 | 广东昭泰体内生物医药科技有限公司 | Method for reprogramming ITNK cells based on CRISPR/Cas9 |
-
2020
- 2020-09-09 CN CN202010942307.9A patent/CN112063653A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120282646A1 (en) * | 2009-10-26 | 2012-11-08 | Korea Research Institute Of Bioscience And Biotechnology | Method for activating a natural killer cell by adjusting the expression of the socs2 gene |
| CN104928242A (en) * | 2015-05-21 | 2015-09-23 | 武汉汉密顿生物科技股份有限公司 | Culturing method of NK (natural killer) cell |
| CN108220235A (en) * | 2016-12-22 | 2018-06-29 | 细胞邦(北京)生物技术有限公司 | A kind of Activated in Vitro expands people's natural kill（NK）The method of cell and its special cultivating system |
| CN109161527A (en) * | 2018-09-25 | 2019-01-08 | 深圳市五零生命科技有限公司 | A kind of efficient NK methods for cell expansion |
| CN109810944A (en) * | 2019-01-24 | 2019-05-28 | 安徽瑞达健康产业有限公司 | A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell |
| CN111607569A (en) * | 2020-06-01 | 2020-09-01 | 广东昭泰体内生物医药科技有限公司 | Method for reprogramming ITNK cells based on CRISPR/Cas9 |
Non-Patent Citations (3)
| Title |
|---|
| MEGAN A. COOPER ET AL.: "Cytokine-induced memory-like natural killer cells", 《PNAS》 * |
| 刁爱坡等: "肿瘤免疫细胞治疗研究进展", 《天津科技大学学报》 * |
| 骆群等: "抗CD28抗体协同刺激增强抗CD3抗体体外激活Ｔ淋巴细胞并降低TGF-β的表达", 《中国实验血液学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574952A (en) * | 2019-09-30 | 2021-03-30 | 英基生物医药(香港)有限公司 | Engineered human immune cell, preparation method and application thereof |
| WO2023038475A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Method for producing directly reprogrammed natural killer cells and use thereof |
| KR20230038629A (en) * | 2021-09-10 | 2023-03-21 | 한국생명공학연구원 | Method for producing directly reprogrammed natural killer cells and uses thereof |
| KR102604208B1 (en) * | 2021-09-10 | 2023-11-23 | 한국생명공학연구원 | Method for producing directly reprogrammed natural killer cells and uses thereof |
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