Cell, immunotherapy product, gene editing method, cell preparation method and application

A gene editing and cell technology, which is applied in the field of immunity and biology, can solve the problems of poor curative effect, tolerance of toxic and side effects, and production cycle and cost of resistant process cells, so as to overcome allogeneic immune rejection and improve tumor immunosuppression. Good effect on environment and safety

Pending Publication Date: 2021-10-01
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the deepening of research and increasing applications, the adverse reactions of CAR-T cell drugs have gradually been recognized and paid attention to, such as the toxic side effects of CAR-T cell therapy (cytokine storm and neurotoxicity), and the adverse effects of solid tumors on them. Tolerance and resistance, tumor immunosuppressive microenvironment, process complexity, cell production cycle and cost, etc. have become bottlenecks restricting CAR-T cell therapy
It is particularly important to actively search for other immunotherapies that can address the shortcomings of current CAR-T cell drugs, and to overcome the challenge of current CAR-T cell ineffectiveness against solid tumors

Method used

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  • Cell, immunotherapy product, gene editing method, cell preparation method and application
  • Cell, immunotherapy product, gene editing method, cell preparation method and application
  • Cell, immunotherapy product, gene editing method, cell preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of lentiviral vector

[0046] Synthesize the ScFv(Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2). 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 EF-1α promoter. The vector was transformed into Stbl3 Escherichia coli strain, screened with ampicillin, and positive clones were obtained, plasmids were extracted, clones were identified by enzyme digestion, and PRRLSIN-ScFV (anti ROBO1-FN3) lentiviral transfection vector ( figure 1 shown).

Embodiment 2

[0047] Example 2 Preparation of lentivirus

[0048] (1) 12 hours before transfection, use about 8×10 per dish 6 293T cells were seeded into 15cm culture dishes. Make sure that the cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

[0049] (2) Prepare solution A and solution B

[0050] Solution A: 6.25ml 2×HEPES buffer.

[0051] Solution B: Add the 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: 6.25 ml.

[0052] Mix solution B thoroughly, and while vortexing solution A gently, add solution B drop by drop and let stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, add dropwise to the culture dish containing 293T cells, gently shake the culture dish back and forth to make the mixture of DNA and calcium ions evenly distributed. ...

Embodiment 3

[0053] Example 3 Preparation of ROBO1 CAR-NK cells

[0054] Adjust the NK-92 cell density to 2-3 x 10 5 / ml, according to the volume ratio (V / V) virus vector: cell culture medium = 1:5, add the virus vector, and add polybrene 8 μg / ml 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. Replenish fluid every 1-2 days to maintain cell density at 2-3×10 5 / ml. After 72 hours, CAR-labeled antibody staining was performed, and ROBO1 CAR NK-92 positive cells were sorted by flow cytometry and expanded for culture. Observe the pH value of the medium, cell density, and cell viability every day and make corresponding records.

[0055] The positive rate of CAR NK-92 cells was detected by flow cytometry, and the results of flow cytometry were as follows: figure 2 shown. figure 2 The antibody use...

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Abstract

The invention relates to a cell, an immunotherapy product, a gene editing method, a cell preparation method and the application. The invention discloses a cell which is a CAR-NK cell obtained after a target site is knocked out through an RNP compound, the RNP compound comprises Cas9 protein and sgRNA, the target site is PD-1, and the sgRNA at least comprises a nucleotide sequence SEQ ID NO: 8 and a nucleotide sequence SEQ ID NO: 12. According to the scheme, the anti-tumor activity of cells and the killing effect on tumor cells are effectively improved, and the compound can be developed into an effective anti-tumor biological preparation.

Description

technical field [0001] The present invention relates to biology and immune technology, in particular to a kind of cell, NK cell immunotherapy product, gene knockout method, cell preparation method and application. Background technique [0002] Tumor is one of the most important diseases that endanger human health. The incidence of tumors has been showing a continuous growth trend, and has become a serious social burden. So far, the development of tumor therapy has experienced breakthroughs from surgical resection, radiotherapy, chemotherapy, targeted drug therapy, and cellular immunotherapy. [0003] In recent years, Chimeric Antigen Receptor T-cell immunotherapy (CAR-T) based on chimeric antigen receptor has attracted much attention. As a "living" drug, CAR-T therapy is very different from traditional drug therapy. This therapy allows the patient's T cells to express chimeric antigen receptors through gene transduction, so that they can not only maintain specific recogni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N9/22C07K19/00A61K39/00A61P35/00
CPCC12N5/0646C12N15/1138C12N9/22C07K16/2803C07K16/30C07K14/7051C07K14/70521A61K39/0011A61P35/00C12N2310/20C12N2510/00C07K2319/02C07K2319/03C07K2319/33
Inventor 张骏韩昆昆李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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