CN110863015A - Target cell with low sensitivity to T cell and preparation method and application thereof - Google Patents
Target cell with low sensitivity to T cell and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a target cell with low sensitivity to T cells, a preparation method thereof and a method for evaluating the effectiveness of CAR-T cells in vitro by using the target cell. The method is characterized in that the sensitivity of target cells to CAT-T cells is reduced by knocking down the HLA expression of the surface target cells, and the occurrence of false positive can be effectively reduced when CAR-T cell products are screened and evaluated in vitro, thereby being beneficial to the earlier stage research of CAR-T products.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a target cell with low sensitivity to T cells, a preparation method thereof and a method for evaluating the effectiveness of CAR-T cells in vitro by using the target cell.
Background
At present, the gene-edited immune cells have excellent performance in tumor treatment, and early screening and preclinical curative effect evaluation of gene-edited immune cell therapy products lack the assistance of in-vitro killing results, but we find that most of tumor cells cultured in laboratories are sensitive to immune cells such as T lymphocytes when screening immune cell therapy products, particularly CAR-T cell therapy products, so that the difference between the killing effect of the unedited immune cells and the killing effect of the edited/modified immune cells is not obvious, and even the 'false positive' of nonspecific killing of the non-target tumor cells is caused. Thus, the killing effect of the CAR-T in vitro can be objectively evaluated, and misjudgment or misjudgment is easy to occur.
Therefore, in order to reduce the adverse effect caused by the non-specific killing, the CRISPR/Cas9 gene editing means is used for knocking out the expression of HLA (human leukocyte antigen) on the surface of a target cell, so that the non-specific killing of the CAR-T cell on the target cell (without expressing the corresponding antigen) is expected to be reduced, the specificity of CAR-T cell therapy is improved, and more accurate therapy is realized.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for preparing a target cell with low sensitivity to T cells, which can effectively reduce the occurrence of false positives when screening and evaluating CAR-T cell products in vitro, and a target cell prepared thereby.
In order to achieve the purpose, the invention adopts the following scheme:
one method of preparing target cells that are less sensitive to T cells is to decrease the sensitivity of target cells to T cells by disrupting HLA expression on the surface of the target cells.
To achieve this, we knocked out the B2M gene on the surface of the target cell, B2M is also called β2Microglobulin, a component of MHC molecules, is a Major Histocompatibility Complex (MHC) class I heavy chain-associated serum protein expressed on the surface of virtually all nucleated cells, while HLA is the expression product of MHC.
Further, gene editing technology is adopted to knock out a gene sequence aiming at the nucleotide sequence shown as SEQ ID NO: 1, designing gRNA. Further, the nucleotide sequence of the gRNA is shown in SEQ ID NO: 2, respectively.
Further, the method comprises the following steps:
1) designing and synthesizing an upstream primer and a downstream primer, wherein the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 3-4, and annealing the upstream primer and the downstream primer into double-stranded gRNA;
2) digesting the vector plasmid to construct an expression vector with gRNA;
3) and (3) transducing the target cells with the expression vector obtained in the step 2 to obtain the target cells which are low sensitive to the T cells.
In certain embodiments, CRISPR-Cas9 gene editing technology can be used, and CRISPR-Cas9 is an adaptive immune defense formed by bacteria and archaea during long-term evolution, and can be used for resisting invading viruses and exogenous DNA. The CRISPR-Cas9 gene editing technology is a technology for carrying out specific DNA modification on a target gene, and is also a method used for the leading edge of gene editing at present. The gene editing technology based on the CRISPR-Cas9 shows great application prospect in the application fields of a series of gene therapies, such as blood diseases, tumors and other genetic diseases. Currently, the technical result is applied to the precise genome modification of human cells, zebrafish, mice and bacteria.
Furthermore, the target cell with low sensitivity to T cells prepared by the preparation method is provided.
Further, the target cells are SKOV3, Hela and K562.
The invention also aims to provide a method for evaluating the effectiveness of the CAR-T cells in vitro by using the target cells, and the evaluation method can be used for more truly evaluating the killing effect of the CAR-T cells in vitro and is beneficial to the prior research of CAR-T products.
In order to achieve the purpose, the invention adopts the following scheme:
the method consists in the following steps:
1) isolation of T lymphocytes and preparation of CAR-T cells
2) Preparation of target cells with low sensitivity to T cells
3) Plating the target cells of the step 2) and carrying out lethality detection.
Furthermore, target cells are adherent cells, the lethality is detected by an ACEA xCELLigence RTCA MP instrument, and the cell killing rate is a baseline resistance index-a real-time resistance index.
Further, the target cells are suspension cells,
the invention has the beneficial effects that:
1) the target cells with low sensitivity to T cells prepared by the invention can effectively reduce the occurrence of false positive when the CAR-T cell products are screened and evaluated in vitro;
2) the evaluation method can be used for more truly evaluating the killing effect of the CAR-T in vitro, and is beneficial to the early research of CAR-T products.
Drawings
FIG. 1 shows the sensitivity test of SKOV3 on T cells.
FIG. 2 shows the sensitivity of Hela to T cells.
FIG. 3 is a CAR-T cell sensitivity assay for negative cells K562.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1 knockdown of HLA expression on the surface of target cells
1. Knockdown of target cell surface HLA (human leukocyte antigen) expression by means of gene editing, targeting SEQ id no: 1 and the CDS region of the gene shown in SEQ ID NO: 2, and a gRNA sequence shown in the specification
2. Construction of CRISPR/Cas9 gene knockout expression vector
The following primers were designed and synthesized, and the specific primers were as follows:
primer sg-B2M-3-up: the nucleotide sequence is shown as SEQ ID NO. 3;
primer sg-B2M-3-down: the nucleotide sequence is shown as SEQ ID NO. 4.
Then, two upstream and downstream primers were annealed into three double-stranded gRNA sequences using the sequences shown above as primers, and the reaction system was loaded according to the instructions of the Annealing Buffer for DNA oligonucleotides (5X) (purchased from Biyunnan Co.).
PX330A-1X3, PX330S-2 and PX330S-3 (all available from Addgene plasma) were cleaved with restriction enzymes BbsI (available from NEB), respectively, according to the instructions. And (3) carrying out agarose gel electrophoresis separation on the enzyme digestion product, then recovering DNA fragments, and then respectively connecting the correspondingly annealed primer fragment and the purified vector fragment by T4 ligase (purchased from Promega company) to obtain an expression vector with only one gRNA. After the expression of Escherichia coli, plasmids were extracted using a plasmid extraction kit (Invitrogen corporation), and the specific method is described in the specification of the kit.
Example 2T cell preparation
(1) Isolation of PBMC
Hydroxyethyl starch (Shandong Qidu pharmaceutical Co., Ltd.) in an amount of 20 vol% was added to peripheral blood of healthy donors, and the mixture was mixed well and allowed to stand at room temperature for 30 min. PBMC was isolated using human lymphocyte isolate (Tianjin tertiary Biochemical Co.). The isolated cells were resuspended in fresh 1640 medium (Gibco) containing 10% FBS and placed in a 37 ℃ CO2 incubator for 2h to remove monocytes.
(2) Activation and expansion of T cells
Activated T lymphocytes obtained in activation (1) were coated with CD3(Miltenyi Biotec) and CD28(Miltenyi Biotec). And performing amplification culture with 1640 culture medium containing 10% FBS (Gibco), and adding 100IU/mL IL-2 (Liaoning satellite biological products) for stimulating proliferation.
Example 3 detection of sensitivity to T lymphocytes following HLA knockdown of adherent cells
T lymphocytes #233, #242, #234, #137, #139, #140, #145, #149, #153, #155 and #161 from different donors were separately activated and cultured according to the method of example 1 to evaluate the killing ability of the T lymphocytes against non-knocked-out HLA and knocked-out HLA when cells sensitive to the T lymphocytes when evaluated in an in vitro or in vivo animal experiment, SKOV3, Hela and K562, respectively, were subjected to targeting at a time ratio of 1:1 for a long time (6 hours, 24 hours). Plating cells according to the designed effective target ratio, detecting the killing performance of the T lymphocytes to target cells with different effective target ratios and different treatment times by using an ACEA xCELLigence RTCA MP instrument, and carrying out the experimental steps according to the instrument instruction. The ACEA xCELLigence RTCA MP principle is that the resistance index is taken as data to record tumor cells attached to the bottom of a hole every 15 minutes, and the proliferation or death condition of the attached target cells is judged through the resistance index. The result formula of the resistance index analysis is as follows: cell killing rate-baseline resistance index-real-time resistance index.
The results are shown in FIG. 1: HLA-class i knockout SKOV3 was significantly less sensitive to T lymphocytes than the no knockout ratio when incubated with T lymphocytes at 1: 16 hours and 24 hours. Specific data are shown in table 1 below:
table 1: detection of sensitivity of SKOV3 to T cells
The results are shown in FIG. 2: the HLA-I knockout Hela was significantly reduced in the ratio of sensitivity to T cells to no knockout when incubated with T lymphocytes at 1: 16 hours and 24 hours. The specific data are shown in the following table 2:
table 2: detection of Hela sensitivity to T lymphocytes
The cells with the HLA knocked out can be used for in vivo and in vitro evaluation of cell therapy, and the T lymphocytes can not be killed highly due to the sensitivity to the T lymphocytes, so that the effective result of the therapy is covered.
Example 4 detection of sensitivity to T lymphocytes following HLA knockdown of suspension cells
K562 cells stably expressing firefly luciferase are taken as suspension cell representatives (K562-luc-GFP for short), cells for knocking out HLA are taken as K562-luc-GFP (KO) for short, CAR-T cells with different structures owned by companies are taken as effector cells, and the sensitivity response of the negative cells K562 is verified during CAR-T effectiveness evaluation. Effector and target cells were plated at 16:1 and 4:1 effector to target ratios. Use of
The killing effect is detected by a standard method provided by a Luciferase Assay System (Promega Cat. # E2520) kit, and the killing rate is calculated by the following formula:
the results are shown in FIG. 3: HLA-class I knockout K562 cells incubated with CAR-T at 16:1 and 4:1 effective target ratios showed a significant decrease in the ratio of sensitivity to no knockout to CAR-T cells. Specific data are shown in table 3 below:
table 3: CAR-T cell sensitivity detection to negative cell K562
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
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<110> Chongqing precision BioIndustrial technology research institute Co., Ltd, Chongqing precision Biotechnology Co., Ltd
<120> target cell with low sensitivity to T cell, preparation method and application thereof
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Claims (9)
1. A method for producing a target cell that is less sensitive to T cells, the method comprising decreasing the sensitivity of the target cell to T cells by disrupting HLA expression on the surface of the target cell.
2. The method of claim 1, wherein the gene editing technique is used to knock out a gene sequence directed to a polypeptide as set forth in SEQ id no: 1, designing gRNA.
3. The method of claim 2, wherein the gRNA has a nucleotide sequence set forth in SEQ ID NO: 2, respectively.
4. The method for preparing according to claim 1, characterized in that the method comprises the following steps:
1) designing and synthesizing an upstream primer and a downstream primer, wherein the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 3-4, and annealing the upstream primer and the downstream primer into double-stranded gRNA;
2) digesting the vector plasmid to construct an expression vector with gRNA;
3) and (3) transducing the target cells with the expression vector obtained in the step 2 to obtain the target cells which are low sensitive to the T cells.
5. A target cell having low sensitivity to T cells produced by the production method according to any one of claims 1 to 4.
6. The target cell of claim 5, wherein the target cell is SKOV3, Hela, and K562.
7. A method for assessing the effectiveness of CAR-T cells in vitro using the target cells of claim 5 or 6, characterized in that said method consists in the following steps:
1) isolating T lymphocytes and preparing CAR-T cells;
2) preparing target cells with low sensitivity to T cells;
3) plating the target cells of the step 2) and carrying out lethality detection.
8. The method of claim 7, wherein the target cells are adherent cells, the lethality is measured using an ACEAxCEELLigene RTCA MP instrument, and the cell killing rate is baseline resistivity index-real time resistivity index.
9. The method of claim 7, wherein the target cells are suspension cells,
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107847524A (en) * | 2015-03-27 | 2018-03-27 | 哈佛学院校长同事会 | By the T cell and its preparation and application of modification |
| US20180141992A1 (en) * | 2014-11-06 | 2018-05-24 | President And Fellows Of Harvard College | Cells lacking b2m surface expression and methods for allogeneic administration of such cells |
| CN114164231A (en) * | 2020-09-10 | 2022-03-11 | 华东师范大学 | Method for gene editing target site in cell |
| CN114645021A (en) * | 2020-12-21 | 2022-06-21 | 未来智人再生医学研究院(广州)有限公司 | Pluripotent stem cell expressing targeted CD47 inhibitory factor, derivative and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180141992A1 (en) * | 2014-11-06 | 2018-05-24 | President And Fellows Of Harvard College | Cells lacking b2m surface expression and methods for allogeneic administration of such cells |
| CN107847524A (en) * | 2015-03-27 | 2018-03-27 | 哈佛学院校长同事会 | By the T cell and its preparation and application of modification |
| CN114164231A (en) * | 2020-09-10 | 2022-03-11 | 华东师范大学 | Method for gene editing target site in cell |
| CN114645021A (en) * | 2020-12-21 | 2022-06-21 | 未来智人再生医学研究院(广州)有限公司 | Pluripotent stem cell expressing targeted CD47 inhibitory factor, derivative and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LIANBO SHAO ET AL.: "Knockout of beta-2 microglobulin enhances cardiac repair by modulating exosome imprinting and inhibiting stem cell-induced immune rejection", 《CELLULAR AND MOLECULAR LIFE SCIENCES》 * |
| 李戊玲 等: "利用CRISPR/Cas9技术制备B2M-细胞模型", 《中国细胞生物学学报》 * |
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