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Anti CD70 CAR-T cell and preparation method and application thereof

A cell and lymphocyte technology, applied in the fields of molecular biology and cell biology, can solve problems such as good curative effect, achieve high-efficiency tumor killing activity, and increase the specificity of T cell killing

Active Publication Date: 2019-12-20
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the research on CAR-T cells in solid tumors is still in the stage of animal experiments, but some small-sample clinical trials and case reports have achieved good results

Method used

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  • Anti CD70 CAR-T cell and preparation method and application thereof
  • Anti CD70 CAR-T cell and preparation method and application thereof
  • Anti CD70 CAR-T cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of lentiviral expression vector

[0045] Gene synthesis of SCFV(anti CD70)-CD8 TM -4-1BB-CD3ζ fusion gene sequence, the gene sequence is shown in SEQ ID NO.7, which is connected to the PLV vector through enzyme digestion transformation, and the upstream of the gene is the EP-1a promoter. Transform the DH5α Escherichia coli strain with the vector, screen with ampicillin, obtain positive clones, extract plasmids, identify the clones by enzyme digestion, and obtain PLV-SCFV(antiCD70)-CD8 TM -4-1BB-CD3ζ lentiviral packaging vector, see figure 1 .

Embodiment 2

[0046] Example 2: Preparation of lentivirus

[0047] 24 hours before transfection, use about 8×10 per bottle 6 293T cells were inoculated into T75 culture flasks. Make sure that the cells are about 80% confluent and evenly distributed in the culture flask for lentiviral packaging.

[0048] Prepare Plasmid and Transfection Reagent Dilutions

[0049] 1. Vortex to mix the PEI 40K transfection reagent.

[0050] 2. Prepare 2 centrifuge tubes, and prepare plasmid and transfection reagent dilutions in the following order.

[0051]

[0052] 3. Mix well.

[0053] 4. Add the transfection reagent dilution (centrifuge tube 2) into the plasmid DNA solution (centrifuge tube 1), and mix thoroughly immediately. Note that the join order is very important.

[0054] 5. Incubate the transfection mixture at room temperature for 15-20 minutes.

[0055] 6. Add 1ml of the transfection mixture into the well-prepared 293T cell culture flask, and gently blow and aspirate the medium to mix.

...

Embodiment 3

[0059] Example 3: Preparation of Anti CD70 CAR-T cells

[0060] Take 0.5ml of blood for rapid detection of pathogenic microorganisms to rule out microbial infections such as HBV, HCV, HDV, HEV, HIV-1 / 2, Treponema pallidum and parasites. Peripheral blood mononuclear cells (PBMC) were collected from the patients by an apheresis machine. Configure complete growth medium, add 5% autologous AB or FBS to PBSPBS, IL-2 concentration is 20ng / ml, dilute the isolated PBMC with medium to 2×10 / ml, take 50u1 flow cytometric detection of T cells in PBMC purity. On day 0, configure buffer1, add 1% FBS to PBS, shake the beads for 30s or manually shake up and down for 5min, take out CD3 / CD8beads according to the ratio of beads to T cells 3 to 1, put them in a 1.5ml EP tube, add 1ml Buffer1 Wash the beads, then use a magnet to suck the beadslmin from the EP tube, discard the washing solution, repeat twice, then use the medium to resuspend the beads to the original volume, mix the cells and the...

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Abstract

The present invention belongs to the fields of molecular biology and cell biology, and particularly relates to a CAR-T cell that specifically recognizes CD70 and a preparation method of the CAR-T cell. SCFV (Anti CD70)-CD8<TM>-4-1BB-CD3[zeta] molecules are expressed in T cells. A CD70 antibody is used for construction of CAR-T cells, CD70 molecules are used as a target antigen, and when the constructed CAR-T cells are in contact with tumor cells expressing the CD70 molecules, distance is pulled closest to increase T cell kill specificity. The prepared Anti-CD70 CAR-T cells has more efficient tumor-killing activity and can be used to prepare tumor drugs with high expression of the CD70 molecules.

Description

technical field [0001] The present invention belongs to the fields of molecular biology and cell biology, and specifically relates to a CAR-T cell specifically recognizing CD70 and a preparation method thereof, and its application in medicines for treating diseases related to highly expressed CD70 molecules. Background technique [0002] 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. Chimeric antigen receptor T-cell immunotherapy (CAR-T), with its unique anti-tumor advantages, has become a hot spot in global biotechnology competition. CAR-T cells combine the single-chain variable region (SCFV) of an antibody that can recognize a certain tumor antigen with CD8 TM , 4-1BB, CD3-ζ chain and other intracellular regions are coupled into a chimeric protein, which is transfected into patient T cells cultured in vitro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K39/00A61P35/00
CPCC12N5/0636C07K16/2875C07K14/7051C07K14/70517C07K14/70578C12N15/86A61K39/001117A61P35/00C12N2510/00C07K2317/622C12N2740/15043C07K2319/02C07K2319/33C07K2319/74A61K2039/5158C12N2740/16043C07K2317/73A61K2039/5156A61K35/17
Inventor 郭志刚纪锋刘蕊邬婷李恩洁李露露李梦晗胡志刚
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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