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ROBO1 CAR-NK cell carrying suicide gene as well as preparation method and application of ROBO1 CAR-NK cell

A suicide gene and cell technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problem that patients cannot collect T cells, the cost is high, and patients cannot accept CAR-T immunotherapy, etc. question

Pending Publication Date: 2020-06-12
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although CAR-T therapy has shown great potential, there are obvious limitations
First, the 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
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; in addition, facing the high cost of CAR-T (refer to the two currently marketed companies, Novartis and Kate), these limitations may prevent some patients who are expected to benefit from CAR-T immunotherapy
In addition, CAR-T therapy also has serious toxicity or side effects, including cytokine release syndrome (CRS), neurotoxicity, etc.

Method used

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  • ROBO1 CAR-NK cell carrying suicide gene as well as preparation method and application of ROBO1 CAR-NK cell
  • ROBO1 CAR-NK cell carrying suicide gene as well as preparation method and application of ROBO1 CAR-NK cell
  • ROBO1 CAR-NK cell carrying suicide gene as well as preparation method and application of ROBO1 CAR-NK cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1. Preparation of lentiviral vector

[0110] ScFv(Anti ROBO1-FN3)-CD8(CD8 TM )-4-1BB-CD3ζ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 8, gene sequence is shown in SEQ ID NO: 10) and mutant ScFv (Anti ROBO1-FN3)-CD8 (CD8 TM )-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 9, the gene sequence is shown in SEQ ID NO: 11, and the mutation site GCC can also be GCG). ScFV(Anti ROBO1-FN3)-CD8 TM -4-1BB-CD3ζ fusion gene as an example to illustrate the preparation process of ROBO1 CAR-NK cells, mutant ScFV (Anti ROBO1-FN3)-CD8 TM - The process of preparing ROBO1M CAR-NK cells with 4-1BB-CD3ζ fusion gene is the same.

[0111] Through enzyme digestion, the ScFv(Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence was transformed and ligated into the pRRLSIN vector, and the upstream of the gene was the EF-1α promoter. Transform the Stbl3 E. coli strain with the vector, screen with ampicillin, obtain positive clo...

Embodiment 2

[0112] Example 2. Preparation of lentivirus

[0113] (1) 24 hours before transfection, use about 8×10 per dish 6 Lenti-X-293T cells were inoculated into a 15cm culture dish. Make sure that the cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

[0114] (2) Prepare solution A and solution B

[0115] Solution A: 6.25ml 2×HEPES buffer (the amount packed in 5 large dishes, the effect is the best).

[0116] Solution B is a mixture of 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 B: 6.25ml.

[0117] 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 dropwise to the culture dish containing Lenti-X-293T c...

Embodiment 3

[0118] Example 3. Preparation of ROBO1 CAR-NK cells

[0119] Adjust the NK92 cell density to (2~3)×10 5 Individuals / ml, the virus prepared in Example 2 was added according to the volume ratio (V / V) virus: cell culture medium=1:5, 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 cells / ml to continue the culture. The next day, all the cells were centrifuged, fresh medium was added, and the culture was continued. Replenish fluid every 1-2 days to maintain cell density at (2-3)×10 5 pieces / 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.

[0120] After flow sorting, the positive ROBO1 CAR NK-92 cells were continuously cultured in the GMP workshop, expanded ...

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Abstract

The invention discloses an ROBO1 CAR-NK cell carrying a suicide gene as well as a preparation method and application of the ROBO1 CAR-NK cell. In order to improve the safety and controllability of theCAR-NK therapy, a suicide gene switch element is integrated into a genome through a lentiviral transfection technology on the basis of the current ROBO1 CAR-NK cell to form the CAR-NK with the suicide gene. By adding the suicide gene, the CAR-NK cells can be better controlled, and the clinical safety is further improved.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a NK cell and its preparation method and application, in particular to a ROBO1 CAR-NK cell carrying a suicide gene and its preparation method and application. Background technique [0002] In recent years, immunotherapy based on CAR-T cells has received widespread attention in the industry. As a "living" drug, CAR-T therapy is very different from traditional drugs. First of all, the therapy requires the isolation of T cells from the patient, modification of T cells with chimeric antigen receptors (CAR) in vitro so that they can specifically recognize cancer cells, and then the modified T cells are expanded and reinfused. into the patient's body. For example, the successful application of CD19 CAR-T cells in patients with CD19-positive malignancies demonstrated the feasibility of this approach for cancer immunotherapy (Grupp et al., 2013). Moreover, CAR-T cells targetin...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/12C12N15/867C12N5/10C07K19/00A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C12N15/86C12N5/0646A61P35/00C07K2319/02C07K2319/03C12N2510/00A61K2039/844A61K2039/812A61K2039/82A61K2039/852A61K2039/884A61K2039/828A61K2039/86A61K39/4613A61K39/4631A61K39/464402C07K2317/622C07K14/705C07K14/70503C12N9/6472C12Y304/22062C07K14/71C12N2502/11A61K48/005C12N2740/16043A61K39/001111C07K14/70517C07K14/70578C07K2317/55C12N15/62C12N2740/15043
Inventor 韩昆昆傅剑敏周敏曾凡军王强李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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