Immune cell capable of simultaneously expressing fusion protein and chimeric antigen receptor and application of immune cell
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A technology of chimeric antigen receptors and immune cells, which is applied in the field of immune cells to achieve the effects of small molecular weight, easy preparation, and reduction of tumor microenvironment
Pending Publication Date: 2022-05-24
SHENZHEN FIRST CONDOR BIOSCIENCE CO LTD
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Although CAR-T cell therapy shines in the treatment of hematological malignancies, it is lackluster in the treatment of solid tumor...
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Embodiment 1
[0089] The preparation of CAR-T immune cells includes the following steps:
[0090] S100: Isolation of peripheral blood PBMC and culture of T cells
[0091] Monocytes were isolated from donor peripheral blood, subjected to density gradient centrifugation using ficol, and enriched for T cells using a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043) using conjugated anti-CD3 / anti-CD28 magnetic beads to activate the culture and expansion of T cells; the medium used TexMACS GMP Medium (MiltenyiBiotec, 170-076-309), containing 10% FBS, 2mM L-glutamine, 100IU / ml rhIL2, all cells were placed at 37°C, 5% CO 2 Cultured in a constant temperature incubator to obtain T cells.
[0092] S200: Cell Line Culture
[0093] Cell line expressing Claudin18.2: 293T-CLDN18.2, constructed by self-produced lentivirus infection.
[0094] Cells for packaging: 293T (human embryonic kidney cell line), purchased from ATCC.
[0095] Establishment of a cell line overexpressing Claudi...
Embodiment 2
[0102] Example 2 CAR-T cell detection test
[0103] 1. CAR positive rate detection
[0104]Using the two lentiviral vectors of step S300 above, two types of CAR-T cells were successfully constructed, named CLDN18.2 CAR-T and CLDN18.2-P50 CAR-T, respectively, with T cells not infected with lentivirus as control (NT).
[0105] like Figure 3a and 3b shown, the proliferation rate of three T cells (CLDN18.2 CAR-T, CLDN18.2-P50 CAR-T, and control NT) after 11 days of culture. Depend on Figure 3a It can be seen that in the absence of antigen stimulation, there is no significant difference in the proliferation rate of the three types of cells, and the expansion fold is basically maintained at about 1100-1200; Figure 3b It can be seen that in the case of antigen stimulation, antigen stimulation has a significant effect on the proliferation rate of CLDN18.2-P50 CAR T cells, with an expansion fold of more than 2000, reaching about 2500, which is much greater than that of CLDN18.2...
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Abstract
The invention discloses an immune cell capable of simultaneously expressing a fusion protein of an intracellular truncated TGF-beta receptor and a cytokine intracellular stimulation domain and a chimeric antigen receptor on a cell membrane. The immune cell can enhance the proliferation capacity of the cell, reduce the influence of a tumor microenvironment, enhance the tumor killing activity of the CAR-T cell and reduce the depletion under the condition of receiving antigen stimulation, so that the negative regulation of the tumor microenvironment on the immune cell is weakened, the depletion of the immune cell is reduced, the tumor killing function of the immune cell is enhanced, and the inhibition of the tumor microenvironment on the immune cell is reduced; by combining with cell factor receptors, corresponding JAK and STAT signal channels are activated, the cells are induced to secrete various active substances such as IFN-gamma, perforin and granzyme, the growth of tumor cells is inhibited, the anti-tumor capability is enhanced, the killing function of immune cells is optimized, and the inhibition of a tumor microenvironment on the immune cells is reduced.
Description
technical field [0001] The invention relates to the technical field of biological cells, in particular to an immune cell that can simultaneously express a fusion protein of intracellular truncated TGF-β receptor and cytokine receptor intracellular stimulation domain and a chimeric antigen receptor (CAR). and its applications. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly, especially adoptive cell therapy (ACT), which refers to the isolation of T cells, NK cells and other immune cells from patients, modified and expanded in vitro, and then returned to the patient. Methods of in vivo tumor therapy. [0003] CAR-T and TCR-T are important components of adoptive cell therapy, especially CAR-T therapy, which has shown good results in the field of hematological tumors and achieved a high remission rate. A typical CAR structure consists of three parts. Extracellular scFv that recognizes tumor antigens, hinge and transmembrane domains, int...
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