Anti-EGFRvIII chimeric antigen receptor, encoding gene, recombinant expression vector, construction method of recombinant expression vector, and application

Abstract

The invention discloses an anti-EGFRvIII chimeric antigen receptor, an encoding gene, a recombinant expression vector, a construction method of the recombinant expression vector, and an application. The anti-EGFRvIII chimeric antigen receptor comprises CD8 leader chimeric receptor signal peptide, an EGFRvIII single-chain antibody light chain VL, Optimal Linker C, an EGFRvIII 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-EGFRvIII 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-EGFRvIII 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. ...

Examples

Embodiment 1

[0082] Example 1 Construction of recombinant lentiviral vector

[0083] 1. Materials

[0084] 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;

[0085] 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:

[0086] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.26)

[0087] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0088] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3'

[0089](SEQ ID NO. 28)

[0090] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0091] VL-F: 5'-CCACGCCGCCAGGCCGGATGTTGTGATGACCCAGACTCC-3' (SEQ ID NO.30)

[0092] VL-R...

Embodiment 2

[0181] Example 2 Concentration and detection of recombinant lentiviral vector

[0182] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0183] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0184] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0185] (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;

[0186] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0187] (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;

[0188] (6) Place the balanced centrifuge tubes symmetrically in the ul...

Embodiment 3

[0266] Example 3 Functional detection of recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR-EGFRvIII-OLC

[0267] 1. Cell-level expression detection of CAR gene:

[0268] (1) After the recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, and lvCAR-EGFRvIII-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, if An increase in the transcription level of CAR mRNA indicates that the transcription level of the CAR gene is successfully expressed;

[0269] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, and lvCAR-EGFRvIII-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;

[0270] (3) Infect t...

technical field

[0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to an anti-EGFRvIII 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. ...

Examples

Embodiment 1

[0082] Example 1 Construction of recombinant lentiviral vector

[0083] 1. Materials

[0084] 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;

[0085] 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:

[0086] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.26)

[0087] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0088] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3'

[0089](SEQ ID NO. 28)

[0090] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0091] VL-F: 5'-CCACGCCGCCAGGCCGGATGTTGTGATGACCCAGACTCC-3' (SEQ ID NO.30)

[0092] VL-R...

Embodiment 2

[0181] Example 2 Concentration and detection of recombinant lentiviral vector

[0182] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0183] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0184] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0185] (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;

[0186] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0187] (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;

[0188] (6) Place the balanced centrifuge tubes symmetrically in the ul...

Embodiment 3

[0266] Example 3 Functional detection of recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, lvCAR-EGFRvIII-OLC

[0267] 1. Cell-level expression detection of CAR gene:

[0268] (1) After the recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, and lvCAR-EGFRvIII-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, if An increase in the transcription level of CAR mRNA indicates that the transcription level of the CAR gene is successfully expressed;

[0269] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR-EGFRvIII-CLA, lvCAR-EGFRvIII-CLB, and lvCAR-EGFRvIII-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;

[0270] (3) Infect t...

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