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Anti-CD33 chimeric antigen receptor, coding gene, recombinant expression vector and construction method and application of recombinant expression vector

A chimeric antigen receptor, chimeric receptor technology, applied in receptor/cell surface antigen/cell surface determinant, application, genetic engineering, etc.

Active Publication Date: 2016-08-03
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-CD33 chimeric antigen receptor, coding gene, recombinant expression vector and construction method and application of recombinant expression vector
  • Anti-CD33 chimeric antigen receptor, coding gene, recombinant expression vector and construction method and application of recombinant expression vector
  • Anti-CD33 chimeric antigen receptor, coding gene, recombinant expression vector and construction method and application of recombinant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Construction of recombinant lentiviral vector

[0080] 1. Materials

[0081] 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. ;

[0082] 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:

[0083] EF1α-F: 5'-attcaaaattttatcgatgctccggtgcccgtcagt-3' (SEQ ID NO.26)

[0084] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0085] CD8leader-F: 5'-ggtgtcgtgaggatccgccaccatggccttaccagtgaccgc-3' (SEQ ID NO.28)

[0086] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0087] VL-F: 5'-ccacgccgccaggccggacatccagctgacccagag-3' (SEQ ID NO.30)

[0088] VL-R: 5'-CTTCACTTCCACCTTGGTGC-3' (SEQ...

Embodiment 2

[0176] Example 2 Concentration and detection of recombinant lentiviral vector

[0177] Purify the recombinant lentiviral vector by ultracentrifugation;

[0178] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0179] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0180] (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;

[0181] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0182] (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;

[0183] (6) Place the balanced centrifuge tubes symmetrically in the ultracentrif...

Embodiment 3

[0259] Example 3 Functional detection of recombinant lentiviral vectors lvCAR33-CLA, lvCAR33-CLB, and lvCAR33-OLC

[0260] 1. Cell-level expression detection of CAR gene:

[0261] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR33-CLA, lvCAR33-CLB, and lvCAR33-OLC, collect the cells and use RT-PCR to detect the transcription level of CAR mRNA to verify the expression of CAR gene. If the transcription level of CAR mRNA increases, it means The transcript level of the CAR gene was successfully expressed;

[0262] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR33-CLA, lvCAR33-CLB, and lvCAR33-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, it means The translational expression of the CAR gene was successful;

[0263] (3) Infect the cells with lvCAR33-CLA, lvCAR33-CLB, lvCAR33-OLC and the control virus MOCK ...

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Abstract

The invention discloses an anti-CD33 chimeric antigen receptor, a coding gene, a recombinant expression vector and a construction method and an application of the recombinant expression vector. The anti-CD33 chimeric antigen receptor comprises a CD8leader chimeric receptor signal peptide, a heavy chain VL of a CD33 single-chain antibody, an Optimal Linker C, a light chain VH of the CD33 single-chain antibody, a CD8Hinge chimeric receptor hinge, a CD8Transmembrane chimeric receptor transmembrane domain, a CD137 chimeric receptor co-stimulation factor and a TCR chimeric receptor T-cell activation domain, which are serially connected in sequence. In addition, the invention discloses the coding gene of the anti-CD33 chimeric antigen receptor, the recombinant expression vector and the construction method and the application of the recombinant expression vector. According to the anti-CD33 chimeric antigen receptor, the secretion of cell factors and the in vitro killing effect of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) 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-CD33 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 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 imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/65C12N5/10A61K35/17A61P35/02
CPCA61K35/17A61K38/00C07K14/7051C07K14/70517C07K14/70596C07K16/2896C07K2317/51C07K2317/515C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/65C12N15/86C12N2510/00C12N2740/15043
Inventor 祁伟俞磊欧黄思
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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