Preparation and Application of Universal Targeting CD19 Antigen Chimeric Receptor T Cells

A chimeric receptor, general-purpose technology, applied in the direction of targeting specific cell fusion, receptors/cell surface antigens/cell surface determinants, polypeptides containing positioning/targeting motifs, etc., can solve the side effects , attack, tumor antigen escape and other issues

Active Publication Date: 2019-12-27
西安桑尼赛尔生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also some problems in the CD19-targeted CAR-T therapy: first, there are short-term side effects, mainly cytokine storm and neurotoxicity, and more than half of the patients have the above-mentioned side effects to varying degrees; According to the results of the study, more than half of the patients will relapse after one year of CD19-targeted CAR-T cell therapy. The reasons for relapse include tumor antigen escape and the exhaustion of imported CAR-T cells
[0020] Although general-purpose CAR-T therapy has many advantages, its production and development are much more difficult than autologous CAR-T therapy
One of the most affecting its efficacy is the problem of allogeneic rejection: different individuals have differences in their histocompatibility antigens, which lead to the attack and rejection of the transplant by recipient T cells

Method used

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  • Preparation and Application of Universal Targeting CD19 Antigen Chimeric Receptor T Cells
  • Preparation and Application of Universal Targeting CD19 Antigen Chimeric Receptor T Cells
  • Preparation and Application of Universal Targeting CD19 Antigen Chimeric Receptor T Cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1: PBMC extraction

[0115] Recruit healthy volunteer A (inconvenience to disclose information), without cold and fever symptoms. Professional medical personnel take 100ml of blood from the median vein of the human elbow and put it into the BD anticoagulant blood vessel. After blood collection, the blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum). Take the PBMC separation tube Sepmate-50, carefully add 15ml of Ficoll buffer, then add the blood PBS mixture, carefully add about 30ml to each tube. After centrifuging at 1200g for 10 minutes, quickly pour the supernatant into a new 50ml tube, centrifuge at 200g for 8 minutes, discard the supernatant, add 10ml PBS buffer to resuspend the pellet, discard the supernatant, add 20ml PBS buffer to resuspend, centrifuge and discard After clearing, add 10ml supernatant PBS buffer to resuspend all pellets. To count the resuspended cells, take 10 μl of the suspension and add 10 μl of ...

Embodiment 2

[0118] Example 2: T cell activation

[0119] Take 2.5 ml of the PBMC cells in Example 1, centrifuge at 200 g for 5 minutes, remove the supernatant, and resuspend with 6 ml of X-VIVO-15 medium. Anti-CD3 / anti-CD28 antibody magnetic beads (Life Technology) were resuspended in PBS buffer (containing 2mM EDTA and 1% fetal bovine serum), added to a magnetic pole and allowed to stand for 2 minutes, then the supernatant was carefully discarded. Repeat the above process 4 times. Take the magnetic beads after washing, put 6×10 6 A magnetic bead was added to the PBMC cells, mixed evenly, and placed in a 37-degree incubator for 3 days. After 3 days, the magnetic beads were taken out, and the T cells were first resuspended several times with a pipette gun. Place the cell suspension in the magnetic pole, and after standing for two minutes, discard the magnetic beads on the tube wall. Count again, and the counting results are shown in Table 2.

[0120] Table 2:

[0121] Cell ...

Embodiment 3

[0122] Example 3: Adeno-associated virus packaging

[0123] (1) Adeno-associated virus vector construction:

[0124] In the present invention, targeting the CD19 CAR gene to knock into the TRAC gene locus adopts the method of CRISPR-Cas9 technology combined with adeno-associated virus vector transduction. Firstly, CRISPR-Cas9 is used to make a cut at the TRAC gene site, and then the CAR gene template delivered by adeno-associated virus is used to repair the gap by homologous recombination, and finally the CAR gene is knocked into the target site.

[0125] In the present invention, the B2M-HLA-E fusion gene is knocked into the B2M gene locus using the method of CRISPR-Cas9 technology combined with adeno-associated virus vector transduction. Firstly, CRISPR-Cas9 is used to create a nick on the gene locus of B2M, and then the B2M-HLA-E fusion gene template delivered by adeno-associated virus is used to repair the gap by homologous recombination, and finally the B2M-HLA-E gene is...

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Abstract

The invention relates to a preparation and an application of universal targeted CD19 antigen chimeric receptor T cells. Concretely, the invention relates to a method for preparing targeted CD19 antigen chimeric receptor T cells. The method comprises delivering a gene editing substance to the T cell to shear TRAC and B2M genes; and cloning the targeted CD19 CAR and B2M-HLA-E fusion gene into a repair template vector to simultaneously introduce targeted CD19 CAR and B2M-HLA-E fusion gene homologous recombination repair templates into T cells. The universal targeted CD19 antigen chimeric receptorT cell has the advantages of small allograft rejection reaction, high yield, high safety, immediate availability, wide application range and the like.

Description

technical field [0001] The invention relates to the field of cell therapy, in particular to the field of cell therapy including general CD19 antigen-targeting chimeric receptor T cells. Background technique [0002] CAR-T therapy [0003] Traditional tumor treatment drugs include chemotherapy drugs and targeted drugs. Although they have improved the survival period of cancer patients to a certain extent, they have also brought serious side effects and greatly reduced the quality of life of patients. Even more unfortunately, most patients will still relapse after receiving these traditional treatments, and once relapse, there is a high probability that there is no cure. [0004] In recent years, with the development of immunotherapy, the emergence of immune checkpoint inhibitor drugs (such as CTLA-4, PD-1 / PD-L1 antibody) has completely changed the way of tumor treatment. However, the effective rate of such drugs in different cancer patients is only 20%-40%, and there are s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/864A61K35/17A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C07K14/70539C12N15/113C12N15/86A61K35/17A61P35/00A61P35/02C12N2510/00C12N2310/10C12N2310/20C12N2750/14143C07K2319/02C07K2319/33C07K2319/00C07K2317/622
Inventor 彭作翰李玏
Owner 西安桑尼赛尔生物医药有限公司
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