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Nucleotide sequence encoding CAR (Chimeric Antigen Receptor), ROBO1 CAR-NK cell expressing CAR as well as preparation and application of ROBO1 CAR-NK cell

A nucleotide sequence and encoding technology, applied in the field of ROBO1CAR-NK cells, can solve the problems of increasing the difficulty of clinical risk operation, endangering the life of patients, on-target/off-target toxicity and neurotoxicity, etc., so as to improve anti-tumor performance and effectively kill tumors , improve the effect of targeting

Active Publication Date: 2019-05-28
四川阿思科力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It mainly uses T-cells as carriers, but when CAR-T cells treat tumors, cytokines such as IL-6 will increase sharply, resulting in cytokine storm phenomenon, and there are problems such as on-target / off-target toxicity and neurotoxicity, and in severe cases, it will life-threatening
Also, T cells must be isolated outside the body (a time-consuming and expensive process)
Moreover, since T cells are modified for specific patients, some patients may not be able to collect T cells, or may not have enough time to wait for the T cell preparation process. Although CAR-T is currently developing towards a general-purpose CAR-T, in fact Increased clinical risk and operational difficulty
In addition, in the face of the high cost of CAR-T, these limitations may prevent some patients who are expected to benefit from CAR-T immunotherapy

Method used

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  • Nucleotide sequence encoding CAR (Chimeric Antigen Receptor), ROBO1 CAR-NK cell expressing CAR as well as preparation and application of ROBO1 CAR-NK cell
  • Nucleotide sequence encoding CAR (Chimeric Antigen Receptor), ROBO1 CAR-NK cell expressing CAR as well as preparation and application of ROBO1 CAR-NK cell
  • Nucleotide sequence encoding CAR (Chimeric Antigen Receptor), ROBO1 CAR-NK cell expressing CAR as well as preparation and application of ROBO1 CAR-NK cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] The preparation of embodiment 1 lentiviral vector

[0104] SCFV (Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 1, gene sequence is shown in SEQ ID NO: 2) and mutant SCFV (Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence were synthesized respectively ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 3, and the gene sequence is shown in SEQ ID NO: 4). Taking SCFV(Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene as an example to illustrate the preparation process of ROBO1 CAR-NK cells, preparation of mutant SCFV(Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene The process of ROBO1M CAR-NK cells is the same.

[0105] Through enzyme digestion, the SCFV (Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence was transformed and ligated into the PRRSLIN vector, and the upstream of the gene was the EP-1α promoter. Transform the Stbl3 E. coli strain with the vector, screen with ampicillin, o...

Embodiment 2

[0106] The preparation of embodiment 2 lentiviruses

[0107] (1) 24 hours before transfection, use about 8×10 per dish 6 293T cells were seeded into 15cm culture dishes. Make sure that the 293T cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

[0108] (2) Prepare solution A and solution B

[0109] Solution A: 6.25ml 2×HEPES buffer (the amount packed in 5 large dishes, the effect is the best).

[0110]Solution B is a mixture obtained by adding the following plasmids: 112.5 μg PRRLSIN-SCFV (anti ROBO1-FN3) (target plasma); 39.5 μg pMD2.G (VSV-G envelope); 73 μg pCMVR8.74 (gag, pol, tat, rev); 625 μl of 2M calcium ion solution. Total volume of solution A: 6.25ml.

[0111] Mix solution B well, and while vortexing solution A gently, add solution B drop by drop to obtain a mixed solution of A and B, and let it stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, add drop by drop to the culture dish containi...

Embodiment 3

[0112] Example 3 Preparation of ROBO1 CAR-NK cells

[0113] Adjust the NK-92 cell density to 2-3 x 10 5 / ml, according to the volume ratio (V / V) virus: cell culture medium = 1:5, the virus prepared in Example 2 was added, and polybrene 8 μg / ml was added at the same time. After 4 h, add an equal amount of fresh complete medium to adjust the cell density to 1×10 5 / ml to continue the culture. The next day, all the cells were centrifuged, fresh medium was added, and the culture was continued. Rehydrate every 1-2 days to maintain the cell density at 2-3×10 5 / ml. After 72 hours, CAR antibody staining was performed, and at the same time, ROBO1 CAR NK-92 positive cells were sorted by flow cytometry and expanded for culture. Observe the color change of the medium, cell density, and cell shape every day and make corresponding records.

[0114] After flow sorting, the positive ROBO1 CAR NK-92 cells were continuously cultured in the GMP workshop, expanded to the volume required fo...

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Abstract

The invention provides a nucleotide sequence encoding a CAR (Chimeric Antigen Receptor) and an ROBO1 CAR-NK cell expressing the CAR. The ROBO1 CAR-NK cell provided by the invention specifically killstumor cells by applying an ROBO1 antibody to the construction of a CAR-NK cell which takes an ROBO1 molecule as a target antigen. The ROBO1 CAR-NK cell can be taken as a medicament for treating tumordiseases, is used for treating tumors with high expression of the ROBO1 molecules, is free from undesirable phenomena such as cytokine storm, and provides a novel treatment method for tumors which cannot be treated with traditional surgery, chemotherapy and radiotherapy; moreover, the ROBO1 CAR-NK cell has the advantages of small toxic and side effects, high safety and better killing effect compared with an ROBO1 CAR-T cell.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a nucleotide sequence encoding CAR, ROBO1 CAR-NK cells expressing the CAR, and preparation and application thereof. Background technique [0002] The mammary gland is composed of skin, fibrous tissue, mammary glands and fat. Breast cancer is a malignant tumor that occurs in the epithelial tissue of the mammary gland. 99% of breast cancer occurs in women, and only 1% in men. [0003] The mammary gland is not an important organ to maintain human life activities, and breast cancer in situ is not fatal; but because breast cancer cells lose the characteristics of normal cells, the connections between cells are loose and easy to fall off. Once the cancer cells fall off, the free cancer cells can spread throughout the body with the blood or lymph fluid, forming metastasis, which is life-threatening. At present, breast cancer has become a common tumor that threatens women's physica...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K38/17A61P35/00C12N5/10C12N15/86C12N15/867C07K2319/03C07K14/7051C07K2319/00C07K16/2803A61K2039/505C07K2317/73C07K2317/622A61K2039/812A61K39/464411A61K2239/47A61K2239/49A61K2239/54A61K2239/38A61K39/4613A61K2239/55A61K2239/31A61K39/4631A61K2239/53A61K35/17C07K14/70517C07K14/70578C12N15/62
Inventor 李华顺韩昆昆王保垒任宝永
Owner 四川阿思科力生物科技有限公司
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