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Targeted folate receptor alpha chimeric antigen receptor as well as preparation method and application thereof

A chimeric antigen receptor, folate receptor technology, applied in receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, chemical instruments and methods , can solve the problem of weak activation of NK cells, and achieve the effect of enhancing the killing ability, high killing efficiency and short time.

Pending Publication Date: 2022-01-07
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently prepared CAR-NK is mainly based on the CAR-T structure and directly transplanted into NK cells. The main problem of this method is that the CAR structure designed for T cells is not suitable for NK cells, so Activation of NK cells is weak

Method used

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  • Targeted folate receptor alpha chimeric antigen receptor as well as preparation method and application thereof
  • Targeted folate receptor alpha chimeric antigen receptor as well as preparation method and application thereof
  • Targeted folate receptor alpha chimeric antigen receptor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Sequence design of a chimeric antigen receptor targeting folate receptor α.

[0026] like figure 1 As shown, in the folate receptor α-targeted chimeric antigen receptor (abbreviated as αFR-CAR), the N-terminal of the folate receptor α-binding polypeptide is connected to the C-terminal of the human CD8α signal peptide, and its C-terminal is connected to the human immunoglobulin The N-terminus of the Fc segment of protein G1 is connected, and the C-terminus of the Fc segment of human immunoglobulin G1 is connected with the N-terminus of the hinge region of human CD8α to form the extracellular domain of the chimeric antigen receptor. The C-terminal of the intracellular segment of human 2B4 is connected with the N-terminal of the intracellular segment of human CD3ζ to form the intracellular segment of the chimeric antigen receptor. The N-terminus of the transmembrane segment of human NKG2D is connected to the C-terminus of the hinge region of human CD8α, and the C-termi...

Embodiment 2

[0071] The lentivirus comprising a chimeric antigen receptor targeting folate receptor alpha is obtained. Specifically include the following steps:

[0072] 1. Preparation of lentivirus overexpressing αFR-CAR, the equipment and materials are shown in Table 1 below.

[0073] Table 1 Equipment and materials required for overexpression of lentiviral vectors

[0074]

[0075] 1.2 Packaging of lentivirus:

[0076] Day0: 293T cells in good condition were replaced with trypsin (TrypLE TM Express) digestion, centrifugation, collection, resuspended cells with DMEM medium (containing 10% FBS) to make a cell suspension, and counted the concentration of the cell suspension. 293T cells in 4 × 10 6 Inoculated in a 10 cm Petri dish and cultured overnight.

[0077] Day1: When the cells grow to 90% confluence, replace the fresh medium one hour in advance, transfect according to the Lipofectamine 2000 instructions, take two 1.5ml centrifuge tubes and add 600μl OPTI-MEM medium respectiv...

Embodiment 3

[0083] The host cell can be one of NK-92 cells, primary human NK cells, primary human T cells, primary human macrophages or cytokine-induced killer cells. Taking NK-92 cells as an example to illustrate the construction of NK-92 cells overexpressing αFR-CAR.

[0084] 1. The required materials are shown in Table 2 below:

[0085] Table 2 Construction materials of NK-92 cells overexpressing αFR-CAR

[0086]

[0087] 1.2 NK-92 infected with overexpression virus of αFR-CAR:

[0088] 1.3 Inoculate NK-92 cells in a 12-well plate, 2×10 per well 5 cells (1ml medium per well, interleukin-2 100U / ml).

[0089] 1.4 Add 50 μl of the lentivirus concentrate prepared in Example 2 and 10 μg / ml polybrene to each well.

[0090] 1.5 at 37°C, 5% CO 2 Culture overnight and replace with fresh medium (containing 100U / ml interleukin-2).

[0091] 1.6 αFR-CAR-NK-92 screening can be carried out five days after NK-92 cells are infected:

[0092] 5 μg / ml puromycin was added to the medium of NK-92 ...

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Abstract

The invention discloses a targeted folate receptor alpha chimeric antigen receptor, and belongs to the technical field of biology. The targeted folate receptor alpha chimeric antigen receptor comprises a human CD8 alpha signal peptide, a polypeptide combined with folate receptor alpha, a human immunoglobulin G1 Fc segment, a human CD8 alpha hinge region, a human NKG2D transmembrane segment, a human 2B4 intracellular segment and a human CD3 zeta intracellular segment. The invention also provides a coding nucleotide molecule of the chimeric antigen receptor, an expression vector, a host cell and a preparation method of the chimeric antigen receptor. The invention also relates to a medicine comprising the chimeric antigen receptor and application of the chimeric antigen receptor in preparation of anti-tumor medicines. The targeted folate receptor alpha chimeric antigen receptor disclosed by the invention can enhance the specific recognition capability of NK cells and enhance the killing capability of the NK cells on folate receptor alpha positive colorectal cancer cell lines.

Description

technical field [0001] The invention relates to a chimeric antigen receptor targeting folic acid receptor α, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] With the development of society, environmental pollution is becoming more and more serious, people's lifestyle and eating habits are changing, and the incidence of cancer is also increasing. Among them, colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, its incidence rate ranks third, and its case fatality rate ranks second. The treatment of CRC includes surgical resection, radiotherapy and chemotherapy, etc., but these treatment methods not only cause adverse reactions but also have poor therapeutic effect to a certain extent. However, as immunotherapy has achieved good curative effects in the comprehensive treatment of various tumors, especially the chimeric antigen receptor (CAR) technology that has emerged...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/00A61P35/00
CPCC07K16/28C07K14/7051A61K39/001111A61P35/00C07K2319/02C07K2319/03A61K39/4613A61K39/4631A61K39/464402
Inventor 敖罗权徐祥敖翔
Owner ARMY MEDICAL UNIV
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