Double-targeting chimeric antigen receptor, encoding gene and recombinant expression vector

A chimeric antigen receptor, dual-targeting technology, applied in vectors, antibody medical components, recombinant DNA technology, etc., can solve problems such as non-existence, and achieve the effect of solving negative recurrence, avoiding off-target effects, and improving therapeutic effects.

Pending Publication Date: 2021-08-13
上海帝鹤思医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, at present, there is no use of MUC1 as a target for pancreatic cancer for the in vivo application of chimeric antigen receptor therapy, and there is no related application of combining MSLN and MUC1 to further improve the therapeutic effect of pancreatic cancer

Method used

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  • Double-targeting chimeric antigen receptor, encoding gene and recombinant expression vector
  • Double-targeting chimeric antigen receptor, encoding gene and recombinant expression vector
  • Double-targeting chimeric antigen receptor, encoding gene and recombinant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Constructing a recombinant expression vector

[0071] 1. Plasmid extraction

[0072] 1. The preparation method of LB liquid medium: Weigh 5g liquid medium dry powder into a 500mL Erlenmeyer flask with an electronic balance, add 100mL pure water, seal it with tin box paper, sterilize it in a high-pressure steam sterilizer, and cool to 40 When ℃~50℃, add 0.2% ampicillin at a ratio of 1000:1, mix carefully, and store at 4 degrees.

[0073] 2, the preparation method of LB solid medium: electronic balance takes 5g solid medium dry powder in 500mL Erlenmeyer flask, adds 100mL ultrapure water, after tin foil sealing, carry out sterilization in autoclave, cool to At 40°C to 50°C, add 0.2% ampicillin at a ratio of 1000:1, mix carefully, invert the plate, and after solidification, stick a parafilm and store at 4°C.

[0074] 3. Take out the glycerol bacteria from the -80°C refrigerator. The glycerol bacteria contain pLenti6.2 / V5-GW / lacZ, lentiviral packaging plasmids p...

Embodiment 2

[0134] The packaging of embodiment 2 recombinant expression vectors

[0135] The four-plasmid packaging system was used for lentiviral packaging. The four plasmids are the lentiviral expression plasmid pLenti6.2 TnMUC1-MSLN CAR containing the CAR structure, and the lentiviral packaging plasmids pLP1, pLP2 and pVSVG; the cells are 293T cells.

[0136] The specific implementation steps are as follows:

[0137]1) Plating within 24 hours before transfection: generally select cells with a passage number of less than 3 generations, adjust the cell density according to the cell growth density and state, the 293T cells in a 10cm dish with a good growth state and a growth density of 80% absorb the waste liquid ;

[0138] 2) Slowly add 3mL PBS to the wall to wash the cells;

[0139] 3) After aspirating and discarding the waste liquid, slowly adhere to the wall and add 1 mL of trypsin, and digest in a constant temperature incubator for 2 minutes;

[0140] 4) Add 3 mL of DMEM complete...

Embodiment 3

[0159] The enrichment of embodiment 3 recombinant expression vectors

[0160] l) Preparation of 5X PEG8000NaCl: Weigh 8766g of NaCl; dissolve 50g of PEG8000 in 200mL Milli-Q pure water;

[0161] 2) Add 7.5mL of 5X PEG8000NaCl mother solution for every 30mL of filtered virus initial solution;

[0162] 3) Mix once every 20-30 minutes, for a total of 3-5 times;

[0163] 4) Store overnight at 4°C;

[0164] 5) 4°C, 4000g, centrifuge for 20min;

[0165] 6) Discard the supernatant, let the tube stand for 1-2 minutes, and absorb the residual liquid;

[0166] 7) Add 300 μL PBS to dissolve the lentivirus precipitate for every 30 mL of filtered virus initial solution;

[0167] 8) The collected virus suspension is divided into 50 μL portions and stored in finished tubes. Quickly freeze with crushed dry ice and store at -80°C for later use (avoid repeated freezing and thawing of concentrated virus liquid);

[0168] 9) It can also be concentrated in an ultracentrifuge (pay attention t...

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Abstract

The invention discloses a double-targeting chimeric antigen receptor, which comprises a double-antigen binding region, a first hinge region, a chimeric antigen receptor T cell activation domain and a protein co-expression self-cleavage domain which are sequentially connected in series, and the double-antigen binding region comprises a heavy chain VH and a light chain VL of an MSLN and MUC1 single-chain antibody which are connected in series, and a second hinge region connected with the MSLN and the MUC1 single-chain antibody. The double-targeting CAR-T cell can kill tumor cells expressing double antigens, killing is more accurate and efficient, meanwhile, the off-target effect is reduced or avoided, the side effect of non-specific killing is reduced, and the anti-tumor effect is enhanced.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to a dual-targeting chimeric antigen receptor, encoding gene, recombinant expression vector and application thereof for immunotherapy of malignant tumors. Background technique [0002] Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. About 90% of them are ductal adenocarcinomas originating from the ductal epithelium. The early diagnosis rate of pancreatic cancer is low, and the cure rate is extremely low. It is one of the malignant tumors with the worst prognosis, and the 5-year survival rate is less than 1%. The leading cause of death is metastatic disease caused by pancreatic cancer. Tumor invasion and metastasis are a continuous process mediated by adhesion molecules, extracellular matrix degradation and angiogenesis by proteases. Pancreatic cancer can occur in the head, body, and tail of the pancreas, or i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00A61P1/18A61P11/00A61P15/14
CPCC07K16/3092C07K16/303C07K16/2821C07K14/70517C07K14/7051C07K14/70521C07K14/70578C07K14/523C12N15/86C12N5/0636A61K39/001168A61K39/00117A61K39/001142A61P35/00A61P1/18A61P11/00A61P15/14C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/812A61K2039/852A61K2039/86
Inventor 陈年红史萍陈春洲万国庆姜丽英顾雪锋
Owner 上海帝鹤思医疗科技有限公司
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