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A kind of target cell for testing tcr-t cell killing effect and its preparation method and application

A technology of TCR-T and TCC-3T3, which is applied in the field of cell engineering, can solve the problems of inability to carry out long-term killing experiments, insensitive killing signals, and high killing background, so as to avoid non-specific killing, sensitive killing signals, and low killing background Effect

Active Publication Date: 2021-02-02
BEIJING DCTY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The target cells of conventional killing experiments mostly use human immortalized cells or tumor cells, such as K562 cells, 293T cells, etc. Since these cells are human immortalized cells, many mutant proteins other than the target antigen protein will be expressed on the cell surface , or presented to the cell surface by HLA molecules, which causes effector cells to kill target cells, in addition to the specific killing of target antigens, there is also non-specific killing of other proteins, and the killing background is high. When the effector cells When the proportion of specific T cells TCR-T cells is low, false negative results will appear
In addition, conventional target cells are not sensitive to killing signals, and their self-proliferation speed is too fast, so they cannot be used for long-term killing experiments.

Method used

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  • A kind of target cell for testing tcr-t cell killing effect and its preparation method and application
  • A kind of target cell for testing tcr-t cell killing effect and its preparation method and application
  • A kind of target cell for testing tcr-t cell killing effect and its preparation method and application

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preparation example Construction

[0033] The present invention also provides a method for preparing the target cell TCC-3T3, comprising the following steps: 1) connecting the Caveolin-1 gene and the caspase-3 gene with a connecting peptide to obtain the fusion protein gene of Caveolin-1 and caspase-3; 2. ) transfer the fusion protein gene of Caveolin-1 and caspase-3 into mouse fibroblast-like cells by lentiviral transfection method to obtain CC-3T3 cells expressing the fusion protein; 3) lentiviral transfection method TCR-T Cell target antigen gene and HLA gene were transferred into CC-3T3 cells to obtain target cell TCC-3T3.

[0034] In the present invention, the fusion protein gene of Caveolin-1 and caspase-3 is obtained by linking Caveolin-1 gene and caspase-3 gene with connecting peptide; Methods: Caveolin-1 gene, connecting peptide gene and caspase-3 gene connected in sequence were synthesized as a fusion gene.

[0035] In the present invention, after the fusion protein gene of Caveolin-1 and caspase-3 is ...

Embodiment 1

[0047] 1. Construction of lentiviral transfection experiment

[0048] (1) Recovery culture HEK-293T ( CRL-11268 TM ), pass on 1-2 generations. Cells were collected and counted the day before transfection, diluted to 5 × 10 5 After the cells / mL, spread 20mL onto a 15cm Petri dish and place it in a 37°C, 5% CO2 incubator for culture.

[0049] (2) On the day of transfection, take two 15mL centrifuge tubes and add plasmids and reagents according to Table 1.

[0050] Table 1 Reagents for transfection

[0051]

[0052] (3) Stand at room temperature for 5 minutes.

[0053] (4) Transfer the liquid from tube A to tube B and mix well.

[0054] (5) Stand still at room temperature for 20 minutes.

[0055] (6) Cell replacement: use a pipette to suck out the culture medium, add 10mL DMEM, shake gently, and then suck it up. Add 18mL DMEM to the Petri dish, shake it gently and place it at 37°C with 5% CO2 for culture.

[0056] (7) Pipette the mixture of tubes A and B with a 5mL p...

Embodiment 2

[0096] The TCR-T cell target antigen gene was replaced by the MAGE-A3 gene, and the rest of the steps were consistent with Example 1.

[0097] The result is as Figure 7 As shown, NIH / 3T3 is a cell without genetic modification; CC-3T3 is a cell stably transfected with fusion genes of Caveolin-1 and caspase-3; TCC-3T3 is a cell stably transfected with fusion genes of Caveolin-1 and caspase-3 and HLA-A0201α chain and β2M gene; TCC-3T3-MAGE-A3 is a cell stably transfected with Caveolin-1 and caspase-3 fusion gene, HLA-A0201α chain, β2M gene and MAGE-A3 gene. NTC is untransfected PBMC cells targeting MAGE-A3 TCR; DMF4 is transfected PBMC cells targeting TCR recognizing MAGE-A3 antigen, and DMF4 recognizes MAGE-A3 antigen dependent on HLA-A0201 presentation.

[0098] TCR-T cells can recognize and kill target cells TCC-3T3 with MAGE-A3 gene antigen, but have no killing effect on CC-3T3 cells and NIH / 3T3 cells that do not have MAGE-A3 gene antigen. And NTC cells did not kill TCC-3T...

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Abstract

The invention provides a target cell TCC-3T3 for testing the killing effect of TCR-T cells, belonging to the technical field of cell engineering; the target cell TCC-3T3 expresses TCR-T cell target antigen gene, HLA gene, Caveolin- 1 and caspase‑3 fusion protein gene in mouse fibroblast-like cells. The preparation method of the target cell TCC-3T3: using the lentivirus transfection method to transfer the fusion protein gene of Caveolin-1 and caspase-3 into mouse fibroblast-like cells to obtain CC-3T3 cells expressing the fusion protein; then The TCR-T cell target antigen gene and HLA gene were transferred into CC-3T3 cells by lentivirus transfection method to obtain the target cell TCC-3T3. The target cell TCC-3T3 has low killing background, is sensitive to killing signals, and can accurately reflect the specific killing ability of effector cells.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a target cell for testing the killing effect of TCR-T cells, a preparation method and application thereof. Background technique [0002] The killing function is an important aspect of the body's immune function. There are a variety of target cells with killing functions in the immune system, such as natural killer cells (NK), cytotoxic T cells (CTL), monocytes and macrophages with ADCC function wait. TCR-T cells are T cells genetically engineered by the receptor, and the adoptive immunotherapy method for tumors based on modified T cells can exert a strong anti-tumor effect in vivo by virtue of its high affinity recognition ability for tumor-specific antigens. Tumor immune effect: It is one of the more effective treatments for malignant tumors. Similar to other immunotherapies, its basic principle is to use the patient's own immune cells to eliminate cancer c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/62C12Q1/02
CPCC07K14/70539C07K2319/00C12N5/0656C12N9/641C12N2510/00C12Y304/22056G01N33/5005
Inventor 焦顺昌张嵘张天赋吴子明
Owner BEIJING DCTY BIOTECH CO LTD
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