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Anti-BCMA chimeric antigen receptor, encoding gene, recombinant expression vector and establishing method and application of anti-BCMA chimeric antigen receptor, encoding gene and recombinant expression vector

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: 2016-07-20
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, encoding gene, recombinant expression vector and establishing method and application of anti-BCMA chimeric antigen receptor, encoding gene and recombinant expression vector
  • Anti-BCMA chimeric antigen receptor, encoding gene, recombinant expression vector and establishing method and application of anti-BCMA chimeric antigen receptor, encoding gene and recombinant expression vector
  • Anti-BCMA chimeric antigen receptor, encoding gene, recombinant expression vector and establishing method and application of anti-BCMA chimeric antigen receptor, encoding gene and recombinant expression vector

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-3Gbasic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzymes were provided by Shiao (Shanghai) Biomedical Technology Co., Ltd. ;

[0083] 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:

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

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

[0086] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.28)

[0087] CD8leader-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 ...

Embodiment 2

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

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

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

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

[0177] (3) Take 6 Hitachi40PA ultracentrifuge tubes, spray 70% ethanol on the surface for disinfection, 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;

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

[0179] (5) Cover the metal cover, balance the centrifuge tube together with the metal cover, and use 1XPBS to adjust the weight deviation within the range of 0.02g; (6) Place the balanced centrifuge tube symmetrically in the ultracen...

Embodiment 3

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

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

[0257] (1) After the recombinant lentiviral vectors lvCAR-BCMA-CLA, lvCAR-BCMA-CLB, and lvCAR-BCMA-OLC infect PBMC cells, collect the cells and use RT-PCR to detect the transcription level of CAR mRNA to verify the expression of the CAR gene. An increase in the transcription level 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. If the CAR protein An increase in the 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...

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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 specifically relates to an anti-BCMA chimeric antigen receptor, an encoding 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 research groups have discovered tumor antigens (tμmorantigens), 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. Non-specific ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/65C12N5/10A61K38/17A61K47/48A61P35/00
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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