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CAR-NK with safety switch and preparation method thereof

A safety switch, NY-ESO-1 technology, applied in the field of natural killer cells and their preparation, can solve the problems of poor antigen receptor targeting CAR-NK cells, inability to recognize antibodies, etc., to improve targeting and improve accuracy sexual effect

Inactive Publication Date: 2020-12-15
SHENZHEN LUOHU PEOPLELS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a natural killer cell modified with a chimeric antigen receptor with a safety switch and a preparation method thereof, aiming at solving the problem of poor targeting of antigen receptors and CAR-NK cells in killing malignant tumor cells in the prior art. After the treatment produces corresponding side effects, it cannot be quickly recognized by antibodies

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  • CAR-NK with safety switch and preparation method thereof
  • CAR-NK with safety switch and preparation method thereof
  • CAR-NK with safety switch and preparation method thereof

Examples

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preparation example Construction

[0057] Specifically, the preparation method of the chimeric antigen receptor modified natural killer cells with a safety switch comprises the following steps:

[0058] S01. Synthesizing the above-mentioned gene fragment for expressing the chimeric antigen receptor with a safety switch, cloning it into a lentiviral expression vector, and preparing a lentiviral expression vector containing the gene fragment of the chimeric antigen receptor with a safety switch;

[0059] S02. Packaging a lentiviral expression vector containing the gene fragment of the chimeric antigen receptor with a safety switch to obtain a lentivirus containing the gene fragment of the chimeric antigen receptor with a safety switch;

[0060] S03. Using the lentivirus containing the gene fragment of the chimeric antigen receptor with safety switch to infect the natural killer cells derived from peripheral blood to obtain the natural killer cells modified with the chimeric antigen receptor with safety switch.

...

Embodiment 1

[0120] Construction of lentiviral expression vector

[0121] Step 1: Sequence Synthesis

[0122] Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize the sequence and construct the chimeric antigen receptor lentiviral expression vector with safety switch of the present invention, namely SP-NY-ESO-1scFv-CD8-41BB-DAP12-T2A-SP-EGFRt A lentiviral expression vector, the lentiviral expression vector includes the antigen receptor with a safety switch, and the antigen receptor with a safety switch includes the first signal peptide SP and NY-ESO-1 single-chain antibody sequentially connected , CD8 hinge region and transmembrane domain, 41BB intracellular domain and DAP12-ITAM domain, T2A, second signal peptide SP and transformed epidermal growth factor receptor; and cloned into pUC57 vector to obtain plasmid pUC57-NY- ESO-1 / CAR-EGFRt.

[0123] The CAR structure diagram of the synthesized NY-ESO-1 / scFv-CD8-41BB-DAP12 is as follows figure 1 , 1303bp long.

[0124] Step 2...

Embodiment 2

[0150] Packaging preparation of lentivirus

[0151] The packaging preparation of lentivirus, the specific operation method is as follows:

[0152] (1) Day1: Take 5×10 6 293FT cells (Invitrogen, Cat.No.R700-07), discard the supernatant after centrifugation, and use 10ml 37 ℃ preheated complete medium (D-MEM+10%FBS+2mM L-glutamine+0.1mM Essential amino acids + 1mM sodium pyruvate + 1% P / S) were resuspended, inoculated in a 10cm petri dish, and incubated overnight at 37°C in a 5% CO2 incubator.

[0153] (2) Day2: Discard the culture medium in the culture dish, add 5ml of 10% FBS containing I culture medium (Invitrogen, Cat. No. 31985-062).

[0154] (3) Preparation of the 2000 complex:

[0155] ① Add 1.5ml of serum-free Add 1 culture solution into a 5ml centrifuge tube, add 9μg ViraPower TM Packaging plasmid mix and 3 μg lentiviral expression plasmid, mixed gently.

[0156] Wherein, the lentiviral expression plasmid is the lentiviral expression vector synthesized above ...

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Abstract

The invention provides a gene segment for expressing a chimeric antigen receptor with a safety switch. The gene segment for expressing the chimeric antigen receptor with the safety switch comprises anNY-ESO-1 single-chain gene segment and a modified epidermal growth factor receptor gene segment which are connected in sequence. The modified epidermal growth factor receptor gene segment comprises an extracellular structural domain III base sequence, an extracellular structural domain IV base sequence and a transmembrane structural domain base sequence of an epidermal growth factor receptor. Onthe one hand, the antigen receptor has improved targeting ability to kill malignant tumor cells. On the other hand, after the CAR-NK cells generate corresponding side effects in the treatment process,the CAR-NK cells can be rapidly removed through the ADCC effect of the NK cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a natural killer cell modified with a chimeric antigen receptor with a safety switch and a preparation method thereof. Background technique [0002] Natural killer (Natural Killer, NK) cells are the core component of human innate immunity. They are the third group of lymphocytes juxtaposed with T and B cells. They are derived from hematopoietic stem cells and mature in the bone marrow. It accounts for about 10% to 15% of the total number of lymphocytes in the peripheral blood, and about 3% to 4% in the spleen. It can also appear in the lungs, liver, and intestinal mucosa, but it is rare in the thymus, lymph nodes, and thoracic duct. Compared with T cells, NK cells have many characteristics: innate immune cells, non-specific direct killing of target cells, no need for pre-sensitization by antigens, no need for antibody participation, no MHC restriction, and early immune killing. NK c...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N5/10C12N15/867
CPCC07K14/71C12N15/86C12N5/0636C07K2319/00C07K2319/03C12N2740/15043C12N2510/00
Inventor 刘兵周美龄刘韬
Owner SHENZHEN LUOHU PEOPLELS HOSPITAL
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