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Chimeric antigen receptor for identifying carcino-embryonic antigens and application of chimeric antigen receptor

A technology of chimeric antigen receptor and carcinoembryonic antigen, which is applied in the field of genetic engineering, can solve the influence of no report showing the stability of chimeric antigen receptor cells and the efficacy of CEA solid tumors, and the stability of antigen chimeric receptors is not very good. It can improve the ability to proliferate and kill tumors, improve the ability to remove tumor cells, and achieve high efficiency.

Active Publication Date: 2017-08-11
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is no good treatment plan for solid tumors expressing CEA, and there is no good solution for the stability of antigen chimeric receptor transfection on T cells, and there is no report showing that M5A-scFv binds antigen chimeric receptor The stability of chimeric antigen receptor cells and the effect on the efficacy of CEA solid tumors

Method used

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  • Chimeric antigen receptor for identifying carcino-embryonic antigens and application of chimeric antigen receptor
  • Chimeric antigen receptor for identifying carcino-embryonic antigens and application of chimeric antigen receptor
  • Chimeric antigen receptor for identifying carcino-embryonic antigens and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Construction of chimeric antigen receptor virus containing scFV of M5A, hMN-14, C2-45 and BW431 / 26

[0067] In order to prove that the anti-CEA CAR we screened has more advantages than the currently used anti-CEA CAR and other anti-CEA scFV CARs, it is necessary to simultaneously construct scFV-CARs of different anti-CEA humanized monoclonal antibodies named CEA-CAR- 1. CEA-CAR-2, CEA-CAR-3 and CEA-CAR-4, as follows:

[0068] 1 Synthesis of gene sequences of chimeric antigen receptors targeting carcinoembryonic antigen containing different scFVs

[0069] Synthesize different single chain antibodies ScFv containing leader peptide (also known as signal peptide) (LP for short), anti-human CEA antigen, IgG4 hinge region (IgG4 for short), CD28 transmembrane region or CD8 transmembrane region (TM for short) , whose structure is as figure 1 shown.

[0070] 2 Construction of lentiviral vector expressing chimeric antigen receptor

[0071] The following primers were...

Embodiment 2

[0093] Example 2 scFV and target antigen (CEA) affinity determination

[0094] The aforementioned 4 scFVs labeled with His tag were cloned into plasmid vector and transfected into HEK293 cells. The transfected cells were cultured for 6 days and all supernatants were collected. The supernatants were filtered through a 0.22um filter and purified by nickel column affinity chromatography. The scFV-His in the serum was purified, and the purity of scFV-His reached 95%. The concentration of scFV-His was determined by Bradford; the affinity of different scFVs to CEA was determined by ForteBio The RED96 interaction analyzer (Pall, CA, U.S.) was used for inspection, and the experimental steps were carried out according to the instructions. The anti-human IgG-Fc (AHC) biosensor was purchased from Pall Forté Bio, 1512121 according to the instructions; the concentration of recombinant CEA-Fc was 15ug / ml, and the product number was 11077-H03H-50 from Sino Biological Inc. Incubated in the ...

Embodiment 3

[0095] Example 3 Detection of Chimeric Antigen Receptor Maintenance in T Cells

[0096] 1 Isolation of human peripheral blood mononuclear cells

[0097] Collect about 60ml of peripheral blood with blood collection tubes with anticoagulant, divide into 30ml of 50ml centrifuge tubes, add 7.5ml of hydroxyethyl starch to dilute; settling naturally at room temperature (18-25℃) for about 30min, collect upper plasma, The collected upper plasma was centrifuged at 1400 rpm / min for 15 min; then the precipitate was resuspended with normal saline, added to the lymphocyte separation medium at a volume ratio of 1:1, gradient centrifuged at 400 g / min, and the deceleration rate of the centrifuge was set to 1 After centrifugation, the centrifuge tube is divided into: first layer: plasma layer; second layer: annular milky white lymphocyte layer; third layer: transparent separation liquid layer; fourth layer: red blood cell layer; Take the second white lymphocyte layer and wash it twice with no...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a chimeric antigen receptor for identifying carcino-embryonic antigens and an application of the chimeric antigen receptor. A single-chain fragment variable (scFV) for identifying the carcino-embryonic antigens, a hinge region, a transmembrane region and an intracellular signal domain are sequentially connected to form the chimeric antigen receptor, and the amino acid sequence of the single-chain fragment variable (scFV) for identifying the carcino-embryonic antigens includes M5A-scFV amino acid sequence or amino acid sequence acquired by performing random mutation on M5A-scFV polypeptide. When the chimeric antigen receptor identifies the carcino-embryonic antigens, T lymphocytes can be more stably expressed, the positive rate of a chimeric antigen receptor of a target CEA (carcino-embryonic antigen) can be maintained in the culture process of patient cells, proliferation capacity and tumor killing capacity of CAR-T (chimeric antigen receptor T lymphocytes) can be improved, the chimeric antigen receptor does not have toxic and side effects on confrontation of original negative cells, can be used for targeted treatment of tumors and high in humanization degree, immunogenicity of the CAR can be effectively reduced, and continuity and safety of the CAR-T in human bodies are improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, relates to a chimeric antigen receptor recognizing carcinoembryonic antigen and its application, and also relates to a lentiviral vector and application comprising a chimeric antigen receptor recognizing carcinoembryonic antigen (CEA) scFV. Background technique [0002] The production technology of monoclonal antibodies has gone through three stages: heterologous polyclonal antibodies produced by classical immunization methods; murine monoclonal antibodies produced by cell engineering and human monoclonal antibodies produced by genetic engineering. When mouse-derived monoclonal antibodies are used for disease treatment, and mouse antibodies are directly used for human treatment, the heterogeneity of mouse antibodies will cause human anti-mouse antibody reaction (HAMA), resulting in antibody half-life Short, is quickly cleared in the circulatory system and loses efficacy. Therefore, therapeutic...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/3007C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 不公告发明人
Owner CHONGQING PRECISION BIOTECH CO LTD
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