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Chimeric antigen acceptor DAP12-T2A-CD8a-CD19scfv-NKp44 and application thereof

A DAP12-T2A-CD8, -cd19scfv-nkp44 technology, applied in the field of immune cells, can solve the problems of easy recurrence of disease, drug resistance, limited treatment methods for B-cell malignant hematological tumors, poor long-term prognosis, etc.

Inactive Publication Date: 2019-03-22
NANJING CART MEDICAL TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The incidence of B-cell malignancies in hematological malignancies is increasing year by year, but the treatment methods for B-cell malignancies are still relatively limited. At present, chemotherapy, stem cell transplantation and biological therapy are the main treatments. High complete remission rate (CR), but such diseases are prone to relapse and subsequent treatment resistance, resulting in poor long-term prognosis

Method used

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  • Chimeric antigen acceptor DAP12-T2A-CD8a-CD19scfv-NKp44 and application thereof
  • Chimeric antigen acceptor DAP12-T2A-CD8a-CD19scfv-NKp44 and application thereof
  • Chimeric antigen acceptor DAP12-T2A-CD8a-CD19scfv-NKp44 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, chimeric antigen receptor preparation

[0051] The invention provides an optimized chimeric antigen receptor targeting CD19 antigen. The chimeric antigen receptor of the present invention is composed of the second intracellular conduction domain-T2A-extracellular signal peptide-targeting CD19 antigen binding domain-the first intracellular conduction domain in series. Therefore, viral vectors containing different combinations of stimulation signals need to be constructed separately. In this example, NKp44 is used as the unified structure of the first intracellular conduction domain, and the following four chimeric antigen receptors need to be constructed respectively ( figure 1 ):

[0052] DAP12-T2A-CD8α signal peptide-VL-Linker-VH-NKp44 (CD19CAR-1)

[0053] DAP12-T2A-CD8α Signal Peptide-VH-Linker-VL-NKp44 (CD19CAR-2)

[0054] DAP12-T2A-GM-CSF signal peptide-VL-Linker-VH-NKp44 (CD19CAR-3)

[0055] DAP12-T2A-GM-CSF signal peptide-VH-Linker-VL-NKp44 (CD1...

Embodiment 2

[0101] Embodiment 2, virus infection T cell

[0102] 1. Isolation and activation of T cells and virus infection

[0103] (1) Isolation of human peripheral blood mononuclear cells

[0104] Use a blood collection tube containing anticoagulant to collect about 10ml of peripheral blood, settle naturally at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and centrifuge the collected upper layer of plasma at 5000r / min for 10min at a volume ratio of 1:1 Add to the lymphocyte separation medium (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.), gradient centrifugation, 3000r / min, centrifugation for 30min, after centrifugation, the centrifuge tube is layered from top to bottom: the first layer is the plasma layer ; The second layer is the buffy coat layer of lymphocytes; the third layer is the transparent separation liquid layer; the fourth layer is the red blood cell layer. Aspirate the buffy coat of lymphocytes, wash twice with ...

Embodiment 3

[0109] Example 3. Effect of Virus Infection of CAR-T Cells on Cell Proliferation

[0110] After each group of viruses infected T cells, the T cells were counted every 1-2 days with a complete medium containing 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581. Then observe the growth of T lymphocytes, the results are as follows: image 3 shown. The results showed that after the cells were infected with CAR-expressing viruses, they could still form typical proliferative clonal clusters. By counting the cells and drawing the cell proliferation curves, it could be seen that CD19CAR-1, CD19CAR-2, CD19CAR-3, and CD19CAR-4 proliferated similarly, compared with T cells uninfected with the virus ( image 3 Medium NTD) proliferative ability was slightly weaker.

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Abstract

The invention discloses an optimized chimeric antigen acceptor DAP12-T2A-CD8a-CD19scfv-NKp44 and application thereof. The chimeric antigen acceptor is formed by serially connecting an intracellular second conduction structural domain DAP12, T2A, a CD8a signal peptide, an anti-human CD19 monoclonal antibody FMC63 light-chain and heavy-chain variable region CD19scFv, and an intracellular first conduction structural domain. The chimeric antigen acceptor is used for modifying T lymphocyte, and the modified T lymphocyte (CAR-T cells) can be used for treatment of CD19 positive hematologic malignancy. In a hematologic malignancy killing test, killing capacity of the CAR-T cells to leukemia tumor cells are obviously enhanced, and good safety and anti-tumor activity are shown in the clinical application.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy. The invention utilizes genetic engineering technology to obtain a fusion gene encoding chimeric antigen receptor DAP12-T2A-CD8α-CD19scFv-NKp44, inserts the gene fragment into a lentiviral expression vector, packages it into a lentivirus, and transduces it into human T lymphocytes to make T cells express the chimeric antigen receptor. The present invention also relates to nucleic acids encoding such transmembrane polypeptides, vectors, and immune cells expressing the CAR on their surface for immunotherapy. The chimeric antigen receptor-modified T cells involved in the present invention can target and specifically kill CD19-positive tumor cells, and are used for the treatment of malignant hematological tumors, opening up an effective adoptive immunotherapy strategy for the treatment of cancer path of. Background technique [0002] With the development of tumor immunology theory and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70503C07K14/70517C07K14/7056C07K16/2803C07K2317/622C07K2319/00C07K2319/02C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 王恩秀汪晨张海
Owner NANJING CART MEDICAL TECH LTD
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