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Chimeric antigen receptor and expression gene thereof, double-antigen regulated type T cell modified by chimeric antigen receptor, and application thereof

A technology of chimeric antigen receptors and gene expression, applied to genetically modified cells, cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, etc., can solve poor specificity and side effects of treatment Waiting for questions

Active Publication Date: 2017-12-15
深圳华云生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In summary, traditional chimeric antigen receptors have many side effects and poor specificity.

Method used

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  • Chimeric antigen receptor and expression gene thereof, double-antigen regulated type T cell modified by chimeric antigen receptor, and application thereof
  • Chimeric antigen receptor and expression gene thereof, double-antigen regulated type T cell modified by chimeric antigen receptor, and application thereof
  • Chimeric antigen receptor and expression gene thereof, double-antigen regulated type T cell modified by chimeric antigen receptor, and application thereof

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Experimental program
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preparation example Construction

[0086] see image 3 , a method for preparing chimeric antigen receptor-modified T cells regulated by dual antigens according to one embodiment, comprising the following steps S110-S130.

[0087] S110. Provide a chimeric antigen receptor expression gene, the chimeric antigen receptor expression gene includes a first fusion protein expression gene and a second fusion protein expression gene, the first fusion protein expression gene includes mucin-1 antibody expression linked in sequence gene, notch receptor expression gene and Gal4-VP64 transcription activator protein expression gene, and the second fusion protein expression gene includes Gal4-UAS promoter expression gene and mesothelin antibody expression gene connected in sequence.

[0088] Specifically, the structure of the chimeric antigen receptor (CAR) can be referred to above, and will not be repeated here.

[0089] In one embodiment, the second fusion protein further includes a terminator linked to the mesothelin antibo...

Embodiment 1

[0103] Preparation of chimeric antigen receptor-modified T cells regulated by dual antigens

[0104] Synthesize the first fusion protein expression gene (including the nucleotide sequence shown in SEQ ID No.1, the nucleotide sequence shown in SEQ ID No.2 and SEQ ID No. The nucleotide sequence shown in No. 3. The expression of the first fusion protein is anti muc-1-synNotch-Gal4VP64.

[0105] The expression gene of the first fusion protein was double digested by EcoR I and BamH I, and cloned into the lentiviral vector pLV-IRES-Puro to obtain the first expression vector. The constructed first expression vector EcoR I and BamH I were double digested and identified, and the electrophoresis of the digested product was as follows: Figure 4 shown (where Figure 4 The 2.4kb fragment is the target fragment, and the 8.1kb fragment is the vector itself). The size of the target fragment was in line with expectations, indicating that the vector was successfully constructed.

[0106] S...

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Abstract

The invention relates to a chimeric antigen receptor and an expression gene thereof, a double-antigen regulated type T cell modified by the chimeric antigen receptor, and application thereof. The expression gene of the chimeric antigen receptor comprises a first fusion protein expression gene and a second fusion protein expression gene, wherein the first fusion protein expression gene comprises a mucoprotein-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcription activating protein expression gene which are sequentially connected; the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and an anti-mesothelin expression gene which are sequentially connected. By adopting the innovative design, the chimeric antigen receptor has the advantages that the chimeric antigen receptor can be successfully imported into the T cell to form the T cell modified by the chimeric antigen receptor; the immune reaction cannot be produced until the mucoprotein-1 and the anti-mesothelin signals simultaneously exist, so as to reach the controllable immune reaction of the chimeric antigen receptor, fewer side effects in treatment, and high specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chimeric antigen receptor and its expression gene, a chimeric antigen receptor modified T cell regulated by double antigens and its application. Background technique [0002] Chimeric antigen receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene. It generally consists of an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain. CAR-T, that is, chimeric antigen receptor (CAR) modified T cells. The application of CAR-T cells in cancer treatment has shown excellent efficacy and great potential. For example, Novartis announced the phase 2 clinical data of its CD19-targeting CAR-T (CTL019) in the treatment of refractory and relapsed acute lymphoblastic leukemia (ALL) at the ASH conference on December 5, 2015. Phase 2 data Similar to the phase 1 data published in 2014, the complete remission rate (CR)...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N5/10C07K19/00A61K35/17A61P35/00
CPCA61K35/17C07K14/4705C07K14/705C07K16/30C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N2510/00
Inventor 黄彪陈雪梅刘韬
Owner 深圳华云生物技术有限公司
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