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CAR-CIK transgenic cell and preparation method and application thereof

A technology of transgenic cells and transgenic carriers, which is applied in the field of medical biology, can solve the problems of lack of antigen recognition specificity and limitations, and achieve the effects of shortening the culture cycle, increasing survival time, and saving production costs

Active Publication Date: 2018-10-16
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lack of antigen recognition specificity limits its application (REN X, MA W, LU H, et al. Modification of cytokine-induced killer cells with chimeric antigen receptors (CARs) enhances antigenor immunity to epidermal growth factor receptor (EGFR)- positive malignancies[J].Cancer Immunol Immunother,2015,64(12):1517-29.)

Method used

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  • CAR-CIK transgenic cell and preparation method and application thereof
  • CAR-CIK transgenic cell and preparation method and application thereof
  • CAR-CIK transgenic cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction of CAR-CIK cells.

[0077] see image 3 , the construction method of the CAR-CIK cell described in the present invention is as follows:

[0078] 1. Isolation of PBMCs.

[0079] (1) Take 50ml of fresh peripheral blood from a healthy donor;

[0080] (2) Spray the blood collection bag with alcohol twice and dry it.

[0081] (3) Use a 50ml syringe to suck out the blood cells in the bag and transfer them to a new 50ml tube.

[0082] (4) Centrifuge at 400g for 10 minutes at 20°C.

[0083] (5) Transfer the upper layer of plasma to a new 50ml centrifuge tube, inactivate the plasma at 56°C for 30 minutes, return to room temperature, centrifuge at 2000g for 30 minutes, and take the supernatant into a 50ml centrifuge tube for later use.

[0084] (6) Make up to 50ml with D-PBS(-), tighten the cap, and mix evenly by inversion.

[0085] (7) Take two new 50ml centrifuge tubes and add 15ml Ficoll lymphocyte separation medium to each tube.

[0086] (8) Care...

Embodiment 2

[0115] Example 2 CAR-T cell construction.

[0116] see Figure 4 , the construction method of the CAR-T cell described in the present invention is as follows:

[0117] 1. Isolation of PBMCs.

[0118] (1) Take 50ml of fresh peripheral blood from a healthy donor;

[0119] (2) Spray the blood collection bag with alcohol twice and dry it.

[0120] (3) Use a 50ml syringe to suck out the blood cells in the bag and transfer them to a new 50ml tube.

[0121] (4) Centrifuge at 400g for 10 minutes at 20°C.

[0122](5) Transfer the upper layer of plasma to a new 50ml centrifuge tube, inactivate the plasma at 56°C for 30 minutes, return to room temperature, centrifuge at 2000g for 30 minutes, and take the supernatant into a 50ml centrifuge tube for later use.

[0123] (6) Make up to 50ml with D-PBS(-), tighten the cap, and mix evenly by inversion.

[0124] (7) Take two new 50ml centrifuge tubes and add 15ml Ficoll lymphocyte separation medium to each tube.

[0125] (8) Carefully ad...

Embodiment 3

[0164] Example 3 Pathogen detection and expression detection of CAR-CIK cells and CAR-T cells.

[0165] 1. Endotoxin detection;

[0166] (1), endotoxin working standard is 15EU / branch;

[0167] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0168] (3) Dilution of endotoxin standard substance: Take one endotoxin standard substance, dilute it with BET water in proportion to dissolve into 4λ and 2λ respectively, seal with parafilm, shake and dissolve for 15min; each step of dilution should be mixed in the vortex Mix on the mixer for 30s;

[0169] (4) Adding samples: Take several LAL reagents, add 0.5 ml of BET water to each tube to dissolve, and distribute to several endotoxin-free test tubes, each tube has 0.1 ml. Two of them are negative control tubes, add 0.1ml of BET water;

[0170] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0171] 2 tubes are sample positive control tubes, add 0.1ml sample soluti...

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Abstract

The invention discloses a CAR-CIK transgenic cell. The CAR-CIK transgenic cell includes a chimeric antigen receptor CAR gene targeting a CD19 antigen, a CD56 cell and a CD3 cell, wherein the CAR gene,the CD56 cell and the CD3 cell are all positive in expression. The invention also discloses a preparation method of the CAR-CIK transgenic cell. The preparation method includes the following steps: step 1, isolating a peripheral blood mononuclear cell (PBMC); step 2, activating a CIK cell; step 3, adding a hCAR19 lentivirus transgenic vector targeting the CD19 antigen for transduction and performing CAR-CIK cell induction culture; and step 4, carrying out amplification in vitro on the CAR-CIK cell to obtain the CAR-CIK transgenic cell. The invention also discloses an application of the CAR-CIK transgenic cell in the preparation of tumor cell therapy medicines. Compared with a CAR-T cell, the CAR-CIK transgenic cell is higher in killing efficiency and wider in killing range.

Description

technical field [0001] The invention belongs to the field of medical biology, in particular to a cell, in particular to a CAR-CIK transgenic cell for tumor immunotherapy. In addition, the present invention also relates to the construction method and application of the cell-related vector. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies, including cytokine therapy, monoclonal antibody therapy, adoptive ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783C12N15/867A61P35/00
CPCC12N5/0638C12N15/86C07K14/7051C07K16/2803C07K2319/33C07K2319/02C07K2317/622C12N2510/00C12N2501/24C12N2501/51C12N2501/515C12N2740/15043A61K39/4631A61K39/4611A61K39/464412A61K39/4613A61P35/00C12N5/10C12N15/867
Inventor 康立清余宙赵世民俞磊祁伟
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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