Chimeric antigen receptor and cell expressing same and preparation method and application of chimeric antigen receptor

Abstract

The invention relates to a chimeric antigen receptor, nucleic acid coding the chimeric antigen receptor, a construct containing nucleic acid, an expression vector, a transformed cell and pharmacy application of the chimeric antigen receptor, nucleic acid coding the chimeric antigen receptor, the construct containing nucleic acid, the expression vector and the transformed cell. The chimeric antigen receptor comprises at least one extracellular domain, an optional transmembrane domain and at least one intracellular signal transduction domain, wherein each extracellular domain comprises an SC-FV fragment derived from a heavy chain and a light chain of an MUC1 or PSCA specific monoclonal antibody or gene modification which is conducted on the basis of the domain and an optimal signal peptide, and each intracellular signal transduction domain comprises a immune costimulatory signal combination. According to the transformed cell, the capacity of the transformed cell for killing solid tumor cells in vitro and in vivo is significantly improved and is higher than that of a conventional chimeric antigen receptor with a structure of the second generation.

Description

technical field

[0001] The invention relates to the technical field of tumor cell immunotherapy, in particular to a chimeric antigen receptor, a nucleic acid encoding it, a construct containing the nucleic acid, an expression vector and transformed cells, and their pharmaceutical use. Background technique

[0002] In recent years, the incidence and mortality of lung cancer have been increasing, making it one of the most serious types of cancer worldwide. Especially in China, the incidence of lung cancer has risen rapidly in the past ten years, mainly due to the rapid increase in the number of smokers (mostly men). According to statistics, about 3,000 people die from smoking every day[1]. Lung cancer is mainly divided into two categories: small cell lung cancer (15%) and non-small cell lung cancer, of which non-small cell lung cancer can be further divided into adenocarcinoma (40%), squamous cell carcinoma (30%) and large cell carcinoma (15%). %). Different types of lung ca...

Examples

Embodiment 1

[0038] Embodiment 1 CAR plasmid construction

[0039] 1. Plasmid construction

[0040] 1) Through gene synthesis, respectively synthesize CAR-PSCA, CAR-MUC1, CAR-PSCA-T1, CAR-PSCA-T2, CAR-MUC1-T1, and CAR-MUC1-T2 (gene sequence map as shown in figure 1 shown), the C-terminal of the synthesized gene contains a restriction endonuclease Pme1 restriction site and its protective bases, and the N-terminal contains a restriction endonuclease Spe1 restriction site and its protective bases.

[0041] For the synthetic gene sequence see figure 1 Sequence map, according to SEQ NO.1 (for the DNA sequence of Anti-PSCA-scFV) and SEQ NO.2 (for the DNA sequence of Anti-MUC-1-scFV) and SEQ NO.3 (for the DNA sequence of the TLR1 domain ) and SEQ NO.4 (the DNA sequence of the TLR2 domain), SEQ NO.5 (the DNA sequence of the CD3ζ domain) and SEQ NO.6 (the DNA sequence of the CD28 intracellular domain).

[0042] 2) Obtain synthetic DNA fragments (CAR-PSCA, CAR-MUC1, CAR-PSCA-T1, CAR-PSCA-T2, CAR-...

Embodiment 2

[0061] Example 2 In vitro detection of the killing function of CAR-MUC1-T1 / 2T cells on tumor (lung cancer) cells

[0062] 1) The GFP T (blank control), CAR-MUC1T (negative control), CAR-MUC1-T1T, and CAR-MUC1-T2T cells prepared in Example 1 were mixed with 1×10 4 The tumor cells A549-GL were mixed at the ratio of 2:1, 1:1, 0.5:1, and 0.25:1, and added to 96-well U-shaped plate, with 3 duplicate holes for each group, centrifuged at 250g for 5min, and placed at 37 ℃5%CO 2 Co-culture in the incubator for 18 hours;

[0063] 2) The recognition and killing functions of GFP T, CAR-MUC1T, CAR-MUC1-T1T, and CAR-MUC1-T2T cells on lung cancer cells were compared in vitro. A549-GL human lung adenocarcinoma cell line with luciferase was used as tumor cells.

[0064] 3) Luciferase (Luciferase) quantitative killing efficiency evaluation method: 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone), 100 μl / well of l...

Embodiment 3

[0066] Example 3 In vitro detection of the killing function of CAR-PSCA-T1 / 2T cells on tumor (lung cancer) cells

[0067]1) The GFP T (blank control), CAR-PSCA T (negative control), CAR-PSCA-T1T, and CAR-PSCA-T2T cells prepared in Example 1 were mixed with 1×10 4 The tumor cells A549-GL were mixed at the ratio of 2:1, 1:1, 0.5:1, and 0.25:1, and added to 96-well U-shaped plate, with 3 duplicate holes for each group, centrifuged at 250g for 5min, and placed at 37 ℃5%CO 2 Co-culture in the incubator for 18 hours;

[0068] 2) The recognition and killing functions of GFP T, CAR-PSCA T, CAR-PSCA-T1T, and CAR-PSCA-T2T cells on lung cancer cells were compared in vitro, and the human lung adenocarcinoma cell line A549-GL with luciferase was selected as the tumor cells.

[0069] 3) Luciferase (Luciferase) quantitative killing efficiency evaluation method: 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone),...

technical field

[0001] The invention relates to the technical field of tumor cell immunotherapy, in particular to a chimeric antigen receptor, a nucleic acid encoding it, a construct containing the nucleic acid, an expression vector and transformed cells, and their pharmaceutical use. Background technique

[0002] In recent years, the incidence and mortality of lung cancer have been increasing, making it one of the most serious types of cancer worldwide. Especially in China, the incidence of lung cancer has risen rapidly in the past ten years, mainly due to the rapid increase in the number of smokers (mostly men). According to statistics, about 3,000 people die from smoking every day[1]. Lung cancer is mainly divided into two categories: small cell lung cancer (15%) and non-small cell lung cancer, of which non-small cell lung cancer can be further divided into adenocarcinoma (40%), squamous cell carcinoma (30%) and large cell carcinoma (15%). %). Different types of lung ca...

Examples

Embodiment 1

[0038] Embodiment 1 CAR plasmid construction

[0039] 1. Plasmid construction

[0040] 1) Through gene synthesis, respectively synthesize CAR-PSCA, CAR-MUC1, CAR-PSCA-T1, CAR-PSCA-T2, CAR-MUC1-T1, and CAR-MUC1-T2 (gene sequence map as shown in figure 1 shown), the C-terminal of the synthesized gene contains a restriction endonuclease Pme1 restriction site and its protective bases, and the N-terminal contains a restriction endonuclease Spe1 restriction site and its protective bases.

[0041] For the synthetic gene sequence see figure 1 Sequence map, according to SEQ NO.1 (for the DNA sequence of Anti-PSCA-scFV) and SEQ NO.2 (for the DNA sequence of Anti-MUC-1-scFV) and SEQ NO.3 (for the DNA sequence of the TLR1 domain ) and SEQ NO.4 (the DNA sequence of the TLR2 domain), SEQ NO.5 (the DNA sequence of the CD3ζ domain) and SEQ NO.6 (the DNA sequence of the CD28 intracellular domain).

[0042] 2) Obtain synthetic DNA fragments (CAR-PSCA, CAR-MUC1, CAR-PSCA-T1, CAR-PSCA-T2, CAR-...

Embodiment 2

[0061] Example 2 In vitro detection of the killing function of CAR-MUC1-T1 / 2T cells on tumor (lung cancer) cells

[0062] 1) The GFP T (blank control), CAR-MUC1T (negative control), CAR-MUC1-T1T, and CAR-MUC1-T2T cells prepared in Example 1 were mixed with 1×10 4 The tumor cells A549-GL were mixed at the ratio of 2:1, 1:1, 0.5:1, and 0.25:1, and added to 96-well U-shaped plate, with 3 duplicate holes for each group, centrifuged at 250g for 5min, and placed at 37 ℃5%CO 2 Co-culture in the incubator for 18 hours;

[0063] 2) The recognition and killing functions of GFP T, CAR-MUC1T, CAR-MUC1-T1T, and CAR-MUC1-T2T cells on lung cancer cells were compared in vitro. A549-GL human lung adenocarcinoma cell line with luciferase was used as tumor cells.

[0064] 3) Luciferase (Luciferase) quantitative killing efficiency evaluation method: 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone), 100 μl / well of l...

Embodiment 3

[0066] Example 3 In vitro detection of the killing function of CAR-PSCA-T1 / 2T cells on tumor (lung cancer) cells

[0067]1) The GFP T (blank control), CAR-PSCA T (negative control), CAR-PSCA-T1T, and CAR-PSCA-T2T cells prepared in Example 1 were mixed with 1×10 4 The tumor cells A549-GL were mixed at the ratio of 2:1, 1:1, 0.5:1, and 0.25:1, and added to 96-well U-shaped plate, with 3 duplicate holes for each group, centrifuged at 250g for 5min, and placed at 37 ℃5%CO 2 Co-culture in the incubator for 18 hours;

[0068] 2) The recognition and killing functions of GFP T, CAR-PSCA T, CAR-PSCA-T1T, and CAR-PSCA-T2T cells on lung cancer cells were compared in vitro, and the human lung adenocarcinoma cell line A549-GL with luciferase was selected as the tumor cells.

[0069] 3) Luciferase (Luciferase) quantitative killing efficiency evaluation method: 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone),...

Images