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CAR-T cell preparation for treating breast cancer and preparation method thereof

A cell preparation and breast cancer technology, applied in the field of CAR-T cell preparation for the treatment of breast cancer and its preparation, can solve the problems of drug resistance, conventional treatment of patient recurrence, etc., and achieve simple production, improved survival cycle and cytokine release, The effect of alleviating the inflammatory response

Inactive Publication Date: 2016-06-08
奥思达干细胞有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, comprehensive treatment has improved the therapeutic effect of breast cancer, but there are still a considerable number of patients who relapse or are resistant to conventional treatment

Method used

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  • CAR-T cell preparation for treating breast cancer and preparation method thereof
  • CAR-T cell preparation for treating breast cancer and preparation method thereof
  • CAR-T cell preparation for treating breast cancer and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 CAR-T cell expression vector construction:

[0029] (1) PCR amplification of the target fragment anti-CA153 and HER2 antibodies Use the pCDNA3-scBW431 / 26-hFc plasmid as a template to design primers, and add NheI and SalI restriction endonuclease sites to the 5' and 3' ends respectively, CEAUpNheI Upstream primer 5'-AGGCTAGCATGGGATGGAGCTGTATCAT-3', CA153-HER2DnSalI downstream primer 5'-AGGTCGACGGTATCGATAAGCTTTG-3', the reaction conditions were 95°C pre-denaturation for 5min, 95°C denaturation for 10s, 68°C annealing for 15s, 72°C extension for 30s, 30 cycles, After the amplification is completed, take out 2 μL sample for identification;

[0030](2) Double enzyme digestion LV-EF1α-Luciferase-PGK-FP635-WPRE vector, target fragment CA153-HER2-CD3-2A-GFP vector digestion system: 50 μL: 10×Greenbuffer 5 μL, vector 3 μL, NheⅠ enzyme 1 μL, SalⅠ enzyme 1 μL, H 2 O40 μL; 50 μL target fragment enzyme digestion system: 10×Greenbuffer 5 μL, target fragment recovery produ...

Embodiment 2

[0032] Embodiment 2CAR-T virus packaging;

[0033] A 4-plasmid system is used for virus packaging, and the 4-plasmid system expresses gag / pol, Rev, VSV-G required for viral vector packaging, and artificial chimeric antigen receptors composed of engineering stable single-chain antibodies. The 4 plasmids were scaled for transient transfection. The total mass is 10 μg. Add the above plasmid to a certain volume of sterile water, and then add 100 μl of 2.5 mmol / L

[0034] CaCl 2 , slowly drop 500 μl of 2×HBS solution into the mixture, add the above solution to the culture dish covered with 293T cells, mix gently, and place at 37°C, 5% CO 2 Cultivate in the incubator for 12 hours, add 8 mL of DMEM liquid medium containing 10% FBS, and continue to cultivate for 48 hours. Collect the supernatant of T cells 48 hours after transfection, centrifuge at 4°C, 2000r / min for 5 minutes to remove cell debris, filter the supernatant with a 0.45μm filter and aliquot, and store at -70°C.

Embodiment 3

[0035] The separation and expansion culture of embodiment 3 T cells:

[0036] 1) Pretreatment of raw blood: extract 100ml of peripheral blood from the patient and add 100μL of heparin anticoagulant, and then divide it into four 50ml centrifuge tubes; dilute the blood with normal saline 1:1 and mix it with a pipette;

[0037] 2) Separation by Ficoll method: carefully add the diluted blood into a 50ml centrifuge tube containing 15ml Ficoll lymphocyte separation medium, about 35ml per tube, and centrifuge at room temperature at 2000rpm for 20 minutes;

[0038] 3) Collection and washing of T cells: pipette the cell layer at the junction of the two liquid surfaces into a 50ml centrifuge tube, supplement to 30ml with normal saline, mix well, centrifuge at room temperature 2000rpm for 10 minutes, and wash twice after the cells settle;

[0039] 4) Expansion culture of T cells: adjust the concentration of T cells to 2×10 6 Inoculate each dish into a 100mm petri dish and place at 37°C,...

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Abstract

The invention discloses a CAR-T cell preparation for treating breast cancer and a preparation method thereof. The preparation disclosed by the invention is prepared from CAR-T cells, human serum albumin, IL-6 receptor antagonist Tocilizumab and normal saline in a certain proportion. The chimeric antigen receptor(CAR) prepared by the method disclosed by the invention is mainly composed of an antigen antibody binding region, a transmembrane region, a CD3zeta intracellular signal region and a co-stimulatory signal transmitting region. According to the CAR-T cell preparation for treating breast cancer disclosed by the invention, the CAR-T cells with chimeric breast cancer cell antigen receptors are used for treating breast cancer, the treatment effect is obvious, and the invention fundamentally provides a method for treating breast cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CAR-T cell preparation for treating breast cancer and a preparation method thereof. Background technique [0002] Among the methods of tumor immunotherapy, adoptive immune effector cell therapy has been paid attention to because of its many advantages. Chimeric Antigen Receptor (CAR)-modified T cells (CAR-T), as a new field of adoptive immune cell therapy, has become a research hotspot. The hypofunction of the immune system of cancer patients due to radiotherapy and chemotherapy is an important reason for the recurrence of the disease, and it is also a major problem that plagues the medical field. CAR-T cell therapy is a brand-new biological immunotherapy method. By extracting T lymphocytes from patients, after 10 to 14 days of in vitro culture and transformation, the carrier is integrated into the normal T cell gene sequence to form a chimeric antigen receptor. Somat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P35/00C12N15/867
CPCA61K35/17C12N15/86C12N2740/15043
Inventor 周萱
Owner 奥思达干细胞有限公司
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