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Molecular switch carried immune cell modified by anti-HER2 (Human Epidermal Growth Factor Receptor 2) chimeric antigen receptor and application thereof

A technology of chimeric antigen receptors and molecular switches, applied to cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, immunoglobulins, etc., can solve the problem of poor prognosis and prone to Metastasis and recurrence and other issues, to achieve the effect of stable genome structure, enhanced killing effect, and high safety

Inactive Publication Date: 2017-12-19
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In China, 30% of breast cancer patients are HER2-positive breast cancer patients. These patients have faster tumor cell malignancy, faster disease progression, more prone to metastasis and recurrence, and poor prognosis

Method used

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  • Molecular switch carried immune cell modified by anti-HER2 (Human Epidermal Growth Factor Receptor 2) chimeric antigen receptor and application thereof
  • Molecular switch carried immune cell modified by anti-HER2 (Human Epidermal Growth Factor Receptor 2) chimeric antigen receptor and application thereof
  • Molecular switch carried immune cell modified by anti-HER2 (Human Epidermal Growth Factor Receptor 2) chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of a HER2-specific CAR expression vector carrying a molecular switch

[0029] Demonstration of the CAR module of Anti-HER2 containing molecular switch figure 2 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence).

[0030] Nucleic acid sequence of each module of CAR carrying molecular switch Anti-HER2

[0031] (1) FKBP-F36V nucleic acid artificial sequence (SEQ ID NO.2)

[0032] (2) linker nucleic acid artificial sequence (SEQ ID NO.3)

[0033] (3) Fas nucleic acid artificial sequence (SEQ ID NO.4)

[0034] (4) Artificial sequence of self-cleaving polypeptide T2A nucleic acid (SEQ ID NO.5)

[0035](5) artificial sequence of leader nucleic acid (SEQ ID NO.6)

[0036] (6) Anti-HER2 single chain antibody fragment (scFv) nucleic acid artificial sequence (SEQ ID NO.7)

[0037] (7) CD8 Hinge region nucleic acid artificial sequence (SEQ ID NO.8)

[0038] (8) CD28 transmembrane region nucleic acid artificial sequence (SEQ ID NO.9)

...

Embodiment 2

[0042] Example 2: Lentivirus packaging and titer detection

[0043] The lentiviral packaging cell line 293T was inoculated in a 10cm culture dish containing DMEM+10%FBS, cultured at 37°C and 5% CO2, and transfected after the adherence rate was 70%-80%. The recombinant plasmid (about 10 μg) and empty plasmid (about 10 μg) in Example 1 were co-transfected into 293T cells with the lentiviral packaging plasmid by calcium phosphate transfection method, mixed gently, and placed at 37°C, 5% Cultivate in a CO2 incubator for 12 hours, add 8 mL of DMEM liquid medium containing 10% FBS, and continue to cultivate for 48 hours. After 48 hours, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72 hours, the supernatant was collected, cell debris was removed, and the virus was harvested and then concentrated to obtain the concentrated HER2-CAR virus solution carrying the molecular switch, which was stored in a -70°C low-te...

Embodiment 3

[0044] Example 3: Preparation of HER2 CAR-T cells with molecular switches and detection of tumor cell killing

[0045] After the virus was concentrated, T cells were transfected, and PBMCs were isolated from peripheral blood with TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biotechnology). After T cells were activated, 1×10 6 cells, add the concentrated HER2-CAR virus solution carrying the molecular switch, mix well, MOI=8. One week later, the transfected T cells were detected for relevant target proteins.

[0046] image 3 The expression of the HER2-CAR vector carrying the molecular switch constructed by the present invention on the surface of T cells detected by flow cytometry. The results showed that 29.2% of the cells were positive for GFP (the lentiviral vector itself expressed GFP protein), indicating that the HER2-CAR carrying the molecular switch constructed by the present invention could be expressed on the surface of T cells, and the ...

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Abstract

The invention discloses a molecular switch carried immune cell modified by an anti-HER2 (Human Epidermal Growth Factor Receptor 2) chimeric antigen receptor and application thereof. The molecular switch carried immune cell modified by the anti-HER2 chimeric antigen receptor contains an anti-HER2 safety type chimeric antigen receptor gene, the anti-HER2 safety type chimeric antigen receptor gene comprises a single-chain antibody of HER2 and is linked with the molecular switch, and the single-chain antibody of the HER2 comprises CD8 Leader, an HER2 binding area, a hinge area of CD8, a transmembrane-stimulation structural domain, and a stimulation signal conduction area of CD3zeta. The immune cell disclosed by the invention is high in stability and is higher in safety; moreover, pathogenic hurt to patients caused by an off-target effect or an immune cytokine release syndrome is prevented.

Description

technical field [0001] The invention relates to the field of biological genes, more specifically, an immune cell modified with an anti-HER2 chimeric antigen receptor carrying a molecular switch and its application. Background technique [0002] In tumor treatment methods, in addition to conventional surgery, radiotherapy, and chemotherapy, chimeric antigen receptor (Chimeric Antigen Receptor, CAR) T cell therapy has become a research hotspot because of its many advantages. The principle of CAR-T cell therapy is to modify T cells. The modified T cells can acquire the ability to specifically recognize tumor-associated antigens and attack and kill tumor cells. It can target and kill tumor cells without harming normal cells. , to achieve the purpose of tumor removal. Compared with radiotherapy and chemotherapy, which cause the decline of immune system function, the disease is easy to relapse, and the drug resistance caused by drug treatment, etc., this method has less toxic and...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/32C07K2317/622C07K2319/02C07K2319/33
Inventor 刘明录冯建海姜夕锋马洪华强邦明金海锋万磊韩庆梅刘敏韩国英卢永灿
Owner SHANDONG XINRUI BIOTECH CO LTD
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