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

A chimeric antigen receptor, chimeric receptor technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, receptor/cell surface antigen/cell surface determinant, application, 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-BCMA chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application
  • Anti-BCMA chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application
  • Anti-BCMA chimeric antigen receptor, coding gene, recombinant expression vector and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Construction of recombinant lentiviral vector

[0081] 1. Materials

[0082] 1. Lentiviral backbone plasmid pLenti-3G ​​basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein pEnv-G, HEK293T / 17 cells, homologous recombinase from Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0083] 2. Primers: The primers needed to amplify DNA fragments and target sites are designed according to the primer design principle. The primers are synthesized by Shanghai Biotechnology Company, specifically:

[0084] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO. 26)

[0085] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO. 27)

[0086] CD8 leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO. 28)

[0087] CD8 leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO. 29)

[0088] VH-F: 5'-CACGCCGCCAGGCCGCAGATTCAGCTGGTGCAGAGC-3' (SEQ ID NO. 30)

[0089] VH-R: 5'-GCTGCTCACGGTCAGGGTG-3' (SEQ ID NO. 31)

[0090]CLA-VL...

Embodiment 2

[0173] Example 2 Concentration and detection of recombinant lentiviral vector

[0174] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0175] (1) Dispense the collected supernatant into a 50ml centrifuge tube, centrifuge at 500g for 10min at room temperature to remove cells and large debris;

[0176] (2) Filter the supernatant with a 0.22μm-0.8μm filter;

[0177] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray the surface with 70% ethanol to sterilize, put them on the ultra-clean bench and irradiate them with a UV lamp to sterilize for 30 minutes. It can also be sterilized by high temperature moist heat;

[0178] (4) Dispense 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0179] (5) Cover the metal cap, trim the centrifuge tube together with the metal cap, and use 1XPBS to adjust the weight deviation within the range of 0.02g; (6) Place the trimmed centrifuge tube symmetrically in the ultracentrifugation rot...

Embodiment 3

[0255]Example 3 Functional detection of recombinant lentiviral vectors lvCAR-BCMA-CLA, lvCAR-BCMA-CLB, and lvCAR-BCMA-OLC

[0256] 1. Cell-level expression detection of CAR gene:

[0257] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR-BCMA-CLA, lvCAR-BCMA-CLB, and lvCAR-BCMA-OLC, collect cells and use RT-PCR to detect CAR mRNA transcription levels to verify CAR gene expression. An increase in the transcription level of CAR mRNA indicates that the transcription level of the CAR gene is successfully expressed;

[0258] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR-BCMA-CLA, lvCAR-BCMA-CLB, and lvCAR-BCMA-OLC, collect the cells and detect the expression level of CAR protein by western blot to verify the expression of the CAR gene. An increase in the protein expression level indicates that the translation level of the CAR gene is successfully expressed;

[0259] (3) Infect the cells with lvCAR-BCMA-CLA, lvCAR-BCMA-CLB, lvCAR-B...

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Abstract

The invention discloses an anti-BCMA chimeric antigen receptor, an encoding gene, a recombinant expression vector and an establishing method and application of the anti-BCMA chimeric antigen receptor, the encoding gene and the recombinant expression vector. The receptor comprises a CD8 leader chimeric receptor signal peptide, a BCMA single-chain antibody heavy chain VH, an Optimal Linker C, a BCMA single-chain antibody light chain VL, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane domain, a CD137 chimeric receptor co-stimulatory factor and a TCR chimeric receptor T cell activating domain which are sequentially connected in series. In addition, the invention further discloses the encoding gene and the recombinant expression vector of the anti-BCMA chimeric antigen receptor and the establishing method and application of the encoding gene and the recombinant expression vector. The secretion of cell factors and the cytotoxicity in vitro of CAR-T cells can be remarkably improved, and the clinical treatment effect is outstanding.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and in particular relates to an anti-BCMA chimeric antigen receptor, an encoding gene, a recombinant expression vector (especially a CAR-T transgenic vector based on a replication-defective recombinant lentivirus) and a construction method thereof and application. 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 to attack tumor cells (highly specific cytolysis). This biological process is very complex and is still under investigation. In the 1990s, several research groups have discovered tumor antigens (tμmor antigens), which T lymphocytes can recognize in a major histocompatibility complex (MHC)-dependent manner. [0003] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Nonspecific immunotherapy ma...

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
CPCA61K38/00C07K14/7051C07K14/70521C07K14/70596C07K16/28C07K2317/51C07K2317/515C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/65C12N15/86C12N2510/00C12N2740/15043
Inventor 祁伟俞磊
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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