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Preparation and construction method of CAR-T carrier applied in colon cancer treatment

A technology of colon cancer and carrier, which is applied in the field of biomedicine to achieve the effects of strong proliferation, enhanced killing effect, and enhanced immune function

Pending Publication Date: 2019-04-30
上海美丽人生医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the process of using CAR-T for anti-tumor treatment, the most critical thing is the construction of the carrier. Although there have been many reports on the construction technology of the CAR-T preparation carrier, there is no effective anti-cancer drug at present. PD-L1 interferes with T cells and has a good recognition of colon cancer cells CAR-T therapeutic vector and related research

Method used

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  • Preparation and construction method of CAR-T carrier applied in colon cancer treatment
  • Preparation and construction method of CAR-T carrier applied in colon cancer treatment
  • Preparation and construction method of CAR-T carrier applied in colon cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of therapeutic vectors for CAR-T preparation

[0044] (1) Plasmid extraction:

[0045] Take out the glycerol bacteria containing the lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro and PUC19 plasmid from the -80°C refrigerator respectively, take 5 μL and inoculate them into 5 mL LB liquid medium (AMP resistance), shake on a constant temperature shaker Cultivate for 12-16 hours at 37°C and 250rpm.

[0046] Plasmid extraction was carried out according to the instructions of Tiangen's plasmid mini-extraction kit.

[0047] (2) Digestion, ligation, transformation

[0048] The extracted lentiviral expression plasmids pCDH-CMV-MCS-EF1-Puro and PUC19 plasmids were simultaneously digested with EcoRI and NotI, and the digested products were subjected to agarose gel electrophoresis, and the results were observed and recorded with a gel imager , pCDH-CMV-MCS-EF1-Puro agarose gel electrophoresis results are as follows image 3 shown.

[0049] Use the...

Embodiment 2

[0063] Embodiment 2: the preparation of concentrated virus liquid

[0064] The three-plasmid packaging system was used for lentiviral packaging. The three plasmids are respectively the therapeutic vector plasmid obtained in Example 1, the lentiviral packaging plasmid pMD2G and the vector plasmid PSPAX2, and the cells are 293T cells.

[0065] The specific implementation steps are as follows:

[0066] (1) Plating within 24 hours before transfection: select cells with a passage number of less than 3 generations, adjust the cell density according to the cell growth density and state, and select 293T cells with a growth density of 80% for plating. In this example, a 6 cm dish was selected. ;

[0067] (2) When the cell growth density reaches 60-90% and the cells are in good condition, the virus packaging can be carried out. The virus packaging is carried out according to the ratio of lentivirus packaging mixture and vector plasmid = 3:2, and the required three-plasmid mixture rati...

Embodiment 3

[0078] Example 3: Acquisition of CAR-T cells

[0079] 1. PBMC (peripheral blood mononuclear cell) isolation

[0080] (1) Use a vacuum blood collection tube containing heparin to collect about 6mL of human peripheral fresh blood;

[0081] (2) Dilution: add an equal volume of PBS at room temperature, and gently pipette to mix;

[0082] (3) Adding samples: Take a 50mL centrifuge tube, draw 6mL Ficoll (lymphocyte separation solution) into the centrifuge tube (the volume ratio of Ficoll to blood before dilution is 1:1), tilt the tube at 45°, and put the diluted blood in About 1cm above the Ficoll liquid level, slowly add to the Ficoll along the tube wall;

[0083] (4) Centrifugation: 18-20°C, 2000rpm, 30min. After centrifugation, it is divided into four layers from the bottom of the tube to the liquid surface, which are red blood cell and granulocyte layer, stratified liquid layer, mononuclear cell layer, and plasma layer;

[0084] (5) Recovery: insert the pipette directly into ...

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Abstract

The invention provides preparation and a construction method of a CAR-T carrier applied in a colon cancer treatment. The provided treatment carrier is composed of two parts: a slow virus expression vector Pcdh-CMV-MCS-EF1-Puro and a CAR expression structure. The CAR structure is CD8leader-EpCAMscfv-CD8[alpha]-CD28-CD137-CD3[zeta]-IRES-PD-1mAb. The provided treatment carrier and package plasmids (PSPAX2 and pMD2G) are packed to form a three plasmid package system, the ratio of PSPAX2 to pMD2G is 8:1, the ratio of PSPAX2 and pMD2G to carrier plasmid is 3:2, and the three plasmid package system is used to infect 293T cells to obtain CAR-T cells. The provided CAR-T cell can target, identify and kill colon cancer cells with EpCAM positive expression.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a preparation and construction method of a CAR-T vector used in the treatment of colon cancer. Background technique [0002] Colon cancer is a malignant lesion of the colonic mucosal epithelium under the action of various carcinogenic factors such as environment or heredity, and is one of the common malignant tumors. In recent years, the incidence and mortality of colon cancer in my country are on the rise. At present, the main treatment methods include surgery, chemotherapy, radiotherapy, and traditional Chinese medicine. However, the therapeutic effect of the above methods is limited, and the side effects are relatively large. Although the surgery-based multidisciplinary team (mμltidisciplinary team, MDT) diagnosis and treatment model for colon cancer and immunotherapy are gradually playing an increasingly important role, they cannot achieve satisfactory results. The abi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N15/66C12N15/86C12N2740/15043
Inventor 田晓丽
Owner 上海美丽人生医疗科技有限公司
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