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Anti-cd20 chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application

A technology of chimeric antigen receptors and chimeric receptors, applied in the fields of receptor/cell surface antigen/cell surface determinant, application, genetic engineering, etc.

Active Publication Date: 2019-07-02
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is worth noting that the above differences are only the conclusions obtained from in vitro experiments, and there is no report comparing the second-generation and third-generation CARs in vivo.

Method used

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  • Anti-cd20 chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application
  • Anti-cd20 chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application
  • Anti-cd20 chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of recombinant lentiviral vector

[0082] 1. Materials

[0083] 1. The lentiviral backbone plasmid pLenti-3G ​​basic, the lentiviral packaging plasmids pPac-GP, pPac-R and the membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzyme were supplied by Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0084] 2. Primers: According to the principles of primer design, the primers required for amplifying DNA fragments and target sites are designed. The primers are synthesized by Shanghai Biological Company, specifically:

[0085] EF1α-F: 5'-attcaaaattttatcgatgctccggtgcccgtcagt-3' (SEQ ID NO. 26)

[0086] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0087] CD8 leader-F: 5'-ggtgtcgtgaggatccgccaccatggccttaccagtgaccgc-3' (SEQ ID NO.28)

[0088] CD8 leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0089] VL-F: 5'-cacgccgccaggccggacattgtgctgacccaatctcc-3' (SEQ ID NO.30)

[0090] VL-R: 5'-ACC...

Embodiment 2

[0179] Example 2 Concentration and detection of recombinant lentiviral vector

[0180] Purify the recombinant lentiviral vector by ultracentrifugation;

[0181] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0182] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0183] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray 70% ethanol on the surface to sterilize them, put them on a clean table and irradiate them with ultraviolet light for 30 minutes to sterilize them. It can also be sterilized by high temperature and moist heat;

[0184] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0185] (5) Cover the metal cover, balance the centrifuge tube together with the metal cover, and adjust with 1XPBS to make the weight deviation within 0.02g;

[0186] (6) Place the balanced centrifuge tubes symmetrically in the ultracentr...

Embodiment 3

[0262] Example 3 Functional detection of recombinant lentiviral vectors lvCAR20-CLA, lvCAR20-CLB, lvCAR20-OLC

[0263] 1. Cell-level expression detection of CAR gene:

[0264] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR20-CLA, lvCAR20-CLB, and lvCAR20-OLC, collect the cells and detect CAR mRNA transcription levels by RT-PCR to verify the expression of CAR genes. If the CAR mRNA transcription levels increase, It indicates that the transcription level of the CAR gene is successfully expressed;

[0265] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR20-CLA, lvCAR20-CLB, and lvCAR20-OLC, collect the cells and detect the expression level of CAR protein by western blot to verify the expression of CAR gene. If the expression level of CAR protein increases, then It shows that the translation level expression of CAR gene is successful;

[0266](3) Infect the cells with lvCAR20-CLA, lvCAR20-CLB, lvCAR20-OLC and the control virus MO...

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PUM

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Abstract

The invention discloses an anti-CD20 chimeric antigen receptor, an encoding gene, a recombinant expression vector, a construction method of the recombinant expression vector and an application. The anti-CD20 chimeric antigen receptor comprises CD8 leader chimeric receptor signal peptide, CD20 VL, Optimal Linker C, a CD20 single-chain antibody heavy chain VH, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane domain, a CD137 chimeric receptor co-stimulated factor and a TCR chimeric receptor T cell activating domain which are connected in series. Moreover, the invention discloses an encoding gene of the anti-CD20 chimeric antigen receptor, a recombinant expression vector, a construction method of the recombinant expression vector, and an application. Secretion of cell factors and an in-vitro killing effect of CAR-T cells are obviously improved, and the CAR-T cells have an outstanding clinical treatment effect.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to an anti-CD20 chimeric antigen receptor, a coding gene, a recombinant expression vector (especially a CAR-T transgene vector based on a replication-defective recombinant lentivirus) and a construction method thereof and apply. 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). This biological process is complex and is still under investigation. In the 1990s, several scientific research groups have discovered tumor antigens (tμmor antigens), and T lymphocytes can recognize these tumor antigens in a major histocompatibility complex (MHC)-dependent manner. [0003] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Nons...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/65C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70596C07K16/2887C07K2317/51C07K2317/515C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/65C12N15/86C12N2510/00C12N2740/15043
Inventor 祁伟俞磊
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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