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Lentiviral vector expressing CD40 antibody, and construction method and application of CAR-T cells

A technology of lentiviral vector and construction method, which is applied in the field of tumor immunotherapy, can solve problems such as tumor deterioration, and achieve the effect of promoting tumor growth and inhibiting macrophages

Inactive Publication Date: 2018-11-30
安徽古一生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem solved by the present invention is that macrophages can promote the deterioration of tumors

Method used

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  • Lentiviral vector expressing CD40 antibody, and construction method and application of CAR-T cells
  • Lentiviral vector expressing CD40 antibody, and construction method and application of CAR-T cells
  • Lentiviral vector expressing CD40 antibody, and construction method and application of CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] (1) Construction of CD40-expressing CAR-T cells carrying PSMA targets

[0066] ① Construction of pCDH-Anti-PSMA scFv-CD8αhinge / TM-CD137-CD3ζ and pCDH-Anti-PSMA scFv-CD8αhinge / TM-CD137-CD3ζ-T2A-CD40 vectors:

[0067] The PSMA.CAR gene is composed of the second-generation CAR. The entire PSMA.CAR is composed of the scFv fragment that recognizes the PSMA molecule, the CD8 transmembrane region, the 4-1BB / CD137 fragment, and the CD3ζ fragment; the scFv is the antibody molecular light of Anti-PSMA. chain and heavy chain to form a single-chain molecule;

[0068] CD8αhinge region / Transmembrane (CD8αhinge / TM), CD8α hinge region and transmembrane region, CD8α hinge region is a flexible region containing a large amount of proline, CD8α transmembrane region mainly anchors the extracellular region and intracellular region on the cell membrane , CD8α hinge region and transmembrane region sequence NM_001768 (1301..1507) found in the Genebank database;

[0069] CD137 is a member of t...

Embodiment 2

[0095] (1) Construction of CD40-expressing CAR-T cells carrying EpCAM targets

[0096] ① Construction of pCDH-Anti-EpCAM scFv-CD8αhinge / TM-CD137-CD3ζ and pCDH-Anti-EpCAM scFv-CD8αhinge / TM-CD137-CD3ζ-T2A-CD40 vectors

[0097] The differences between this embodiment and the construction method in Embodiment 1 are:

[0098] The scFv fragment sequence of the EpCAM target is derived from the antibody sequence published by the patent number CN 107602703A, and the specific sequence is shown in SEQ ID NO.6.

[0099] Molecular structure diagram of Anti-EpCAM scFv-CD8αhinge / TM-CD137-CD3ζ-T2A-CD40 Figure 7 shown.

[0100] (2) Functional detection of CAR-T cells expressing CD40 antibody carrying EpCAM target

[0101] ①CAR-T cell proliferation and maintenance ability in vitro:

[0102] After using the lentivirus carrying the CAR gene of the EpCAM target point and the lentivirus carrying the CAR gene of the EpCAM target point of the CD40 antibody to infect the T cells, on days 2, 4, 6,...

Embodiment 3

[0110] (1) Construction of CAR-T cells expressing CD40 antibodies carrying Her2 targets

[0111] ① Construction of pCDH-Anti-Her2scFv-CD8αhinge / TM-CD137-CD3ζ and pCDH-Anti-Her2scFv-CD8αhinge / TM-CD137-CD3ζ-T2A-CD40 vectors

[0112] The difference between this example and the construction method in Example 1 is that the scFv fragment sequence of the Her2 target is derived from the antibody sequence published by the patent number CN 107488636 A, and the specific sequence is shown in SEQ ID NO.7.

[0113] Molecular structure diagram of Anti-Her2scFv-CD8αhinge / TM-CD137-CD3ζ-T2A-CD40 Figure 13 shown.

[0114] (2) Functional detection of CAR-T cells carrying CD40 antibody expressed by Her2 target

[0115] ①CAR-T cell proliferation and maintenance ability in vitro:

[0116] After infecting T cells with the lentivirus carrying the CAR gene of the Her2 target point and the lentivirus carrying the CAR gene of the Her2 target point of the CD40 antibody, on days 2, 4, 6, 8, 10, 12, and...

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Abstract

The invention discloses a lentiviral vector expressing a CD40 antibody, and a construction method and application of CAR-T cells, and relates to the technical field of tumor immunotherapy. The lentiviral vector expressing the CD40 antibody, and the construction method and application of the CAR-T cells are put forward based on the problem that macrophages promote tumor deterioration. The lentiviral expression vector carrying CAR genes of solid tumor specific antigen target spots of the CD40 antibody, and the CAR-T cells expressing the solid tumor specific antigen target spots of the CD40 antibody are included. The lentiviral vector expressing the CD40 antibody, and the construction method and application of the CAR-T cells have the beneficial effects that the adverse effects that T cells for identifying a certain antigen by relying on TCR proliferate in quantity, and inflammation is likely to be caused are solved, and it is ensured that the CAR-T cells can exert the tumor killing capacity specifically and exclusively.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to a lentiviral vector expressing CD40 antibody, a construction method and application of CAR-T cells. Background technique [0002] At the beginning of 2017, the National Cancer Center released the latest cancer data in China, each of which tells us with objective facts: China is facing a war against cancer. About 10,000 people are diagnosed with cancer every day, and about 7 people are diagnosed with cancer every minute. The number of new cancer cases continues to rise, from 3.58 million to 3.68 million, an increase of 3%. The number of new cancer cases in the world is about 14.09 million, and China's new cancer cases account for 1 / 4 of the world's total. The treatment of cancer in our country is facing huge challenges. At present, traditional therapies mainly include surgery, chemotherapy, radiotherapy and targeted therapy, which have certain effects in the treatment...

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
CPCA61K35/17A61P35/00C12N5/0636C12N15/66C12N15/86C12N2740/15043
Inventor 王维韩江萌刘振云程丰伟徐华栋
Owner 安徽古一生物科技有限公司
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