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NK (Natural Killer) cell with chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and preparation method and application thereof

A chimeric antigen receptor, NK cell technology, applied in the biological field, can solve the problems of large demand for cytokines, poor cell expansion effect, and unstable culture effect.

Active Publication Date: 2019-03-26
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, NK in vitro expansion technology is mainly realized by adding cytokines such as IL-2, IL-12, IL-15 and IFN-γ into the in vitro culture system, but the effect of cell expansion is poor and the purity is low, which is harmful to cytokines. The demand is large, and the cultivation effect is unstable

Method used

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  • NK (Natural Killer) cell with chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and preparation method and application thereof
  • NK (Natural Killer) cell with chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and preparation method and application thereof
  • NK (Natural Killer) cell with chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Construction and amplification of CEA-CAR-pLVX plasmid

[0083] 1. Construction of CEA-CAR-pLVX plasmid

[0084] according to figure 2 The shown process constructs the CEA-CAR-pLVX plasmid, the specific construction method is as follows:

[0085] 1. Entrust GenScript Biotechnology Co., Ltd. to synthesize the gene sequence fragments containing restriction sites HindⅢ (-AAGCTT-) and BglⅡ (-AGATCT-) according to the gene sequence SEQ ID NO.1 provided by this research unit, and combine them Clone into the pUC57 plasmid (GenScript Biotechnology Co., Ltd., item number: SD1176), and the resulting recombinant plasmid is the CEA-CAR-pUC57 plasmid.

[0086] The CEA-CAR-pUC57 plasmid is a plasmid obtained by inserting the DNA molecule shown in SEQ ID NO.5 between the HindIII and BglII restriction sites of the pUC57 plasmid.

[0087] 2. The pLVX-IRES plasmid (Taksra company, article number: 631238) and CEA-CAR-pUC57 plasmid were simultaneously digested with Hind III (Taksra comp...

Embodiment 2

[0095] Example 2: Packaging of lentiviral vectors and detection of lentiviral titer

[0096] 1. Packaging of lentiviral vectors

[0097] 1. Resuscitate 293T cells and culture them. The culture medium is DMEM medium (Gibico company, product number: 11995065) containing 10% fetal bovine serum (Gibico company, product number: 10099141). Adjust the cell concentration so that the culture volume of the cells in a 10 cm culture dish is 10 mL and the confluence is 80%.

[0098] 2. According to the method in Example 1, the plasmid gag (Youbao Bio, product number: VT1548), Rev (Youbao Bio, product number: VT1445), VSV-G (Youbao Bio, product number: VT1491) for lentivirus packaging Perform amplification and concentration detection. The results showed that the above plasmid concentrations were 0.8-1.2ug / ul.

[0099] 3. Add 20ug of the gag plasmid, 30ug of Rev plasmid, 15ug of VSV-G plasmid and 35ug of CEA-CAR-pLVX plasmid prepared in Example 1 to 1mL DMEM medium. After mixing, incubate at room ...

Embodiment 3

[0107] Example 3: Preparation of CEA-CAR-NK cells

[0108] 1. Cultivation of NK cells and preparation of CEA-CAR-NK cells

[0109] 1. Use heparin anticoagulation tube to collect 50-80mL peripheral blood intravenously, centrifuge at 1500r / mim for 5min, pipette the upper serum for use, resuspend the cell pellet with the same amount of PBS, and slowly add it to the same volume of Ficoll Saccharide-diatophane meglumine) separation solution (purchased from General Electric Medical Group, article number: 17-5442-02 / 03), set the natural lifting speed of the centrifuge, centrifuge at 2000r / min for 20 minutes, and collect the cells in the middle buffy coat after centrifugation For peripheral blood mononuclear cells.

[0110] 2. Use 5% inactivated autologous serum (preparation method of inactivated autologous serum: place the serum in a water bath at 56°C for half an hour), 500IU / mL IL-2 (Invitrogen, article number: PA5-46923), Culture the peripheral blood mononuclear cells obtained in step ...

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Abstract

The invention discloses a NK (Natural Killer) cell with a chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and a preparation method and applications thereof. The NK cell with the chimeric CEAantigen receptor, which is disclosed by the invention, is obtained by expressing the chimeric CEA antigen receptor in a NK cell. The NK cell with the chimeric CEA antigen receptor has the following characteristics that: 1, tumor cells can be efficiently and specifically killed; 2, while the CEA-CAR (Chimeric Antigen Receptor) is expressed, the regulatory mechanism of the NK cell per se is not affected, that is, the ability to inhibit the effect on homologous normal cells is not affected; and 3, after in vitro expansion of lymphocytes, the NK cell accounts for more than 90% of the harvested total cells and the CEA-CAR-NK accounts for more than 34% of the harvested total cells. The NK cell with the chimeric CEA antigen receptor, which is disclosed by the invention, plays an important role in clinical treatment of various solid tumors when serving as a biological agent.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a chimeric CEA antigen receptor NK cell and its preparation method and application. Background technique [0002] Chimeric angtigen receptor (CAR) is a type of antigen that can be expressed on the cell membrane, and can bind to a specific signal molecule outside the cell to activate a series of biochemical reactions in the cell, so that the cell can specifically recognize external stimuli and Special proteins that produce corresponding effects. CAR is mainly composed of three parts: extracellular antigen binding area, hinge area and intracellular signal transduction area. The extracellular domain of CAR is a single chain Fv domain (scFv), which has the function of specifically recognizing and binding antigen. Its composition is to use the variable region of heavy chain (VH) and the variable region of light chain (VL) of the monoclonal antibody with a flexible polypeptide linker ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/62C12N15/867A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0646C12N15/86C07K14/7051C07K14/70517C07K14/70578C07K16/3007C07K2319/74C07K2319/33C07K2319/00C07K2317/622C12N2740/15043C12N2510/00C12N2800/107
Inventor 卢戌刘静维王跃刘雪松李君阳
Owner BEIJING BIOHEALTHCARE BIOTECH
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