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Chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 and purpose thereof

A DAP12-T2A-CD8, -cd19scfv-trem1 technology, applied in the field of immune cells, can solve the problems of easy relapse of the disease, drug resistance, poor long-term prognosis, and limited treatment methods for B-cell malignant hematological tumors.

Inactive Publication Date: 2019-03-15
NANJING CART MEDICAL TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 and purpose thereof
  • Chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 and purpose thereof
  • Chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 and purpose thereof

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Embodiment 1

[0047] Embodiment 1, chimeric antigen receptor preparation

[0048] 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, TREM1 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 ):

[0049]

[0050] 1. Contains gene sequence targeting CD19 chimeric antigen receptor

[0051] The design sequentially contains natural killer activating receptor (DAP12 for short), T2A, CD8α signal peptide, GM-CSF signal peptide, VL-Linker-VH, VH-Linker-VL, human myeloid cel...

Embodiment 2

[0095] Embodiment 2, virus infection T cell

[0096] 1. Isolation and activation of T cells and virus infection

[0097] (1) Isolation of human peripheral blood mononuclear cells

[0098] 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

[0103] Example 3. Effect of Virus Infection of CAR-T Cells on Cell Proliferation

[0104] 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 can be seen that the proliferation of CD19CAR-1, CD19CAR-2, CD19CAR-3, and CD19CAR-4 were similar, 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 a chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 and a purpose thereof. The chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 comprises an intracellularsecond transduction domain DAP12, T2A, a CD8alpha signal peptide, anti-human CD19 monoclonal antibody FMC63 light chain and heavy chain variable regions CD19scFv and an intracellular first transduction domain which are connected with each other in series. The chimeric antigen receptor DAP12-T2A-CD8alpha-CD19scFv-TREM1 disclosed by the invention is used for modifying T lymphocytes; and the modifiedT cells (CAR-T cells) can be used for treating CD19 positive hematologic malignancies. In a hematologic malignancy killing test, the killing ability of the CAR-T cells to leukemia tumor cells is obviously enhanced, and good safety and antineoplastic activity are shown in clinical application.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy. The present invention uses genetic engineering technology to obtain a fusion gene encoding chimeric antigen receptor DAP12-T2A-CD8α-CD19scFv-TREM1, 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 a...

Claims

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Application Information

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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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