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Chimeric antigen receptor of cell for targeted expression of Claudin 18.2 and application of chimeric antigen receptor

A technology of chimeric antigen receptors and expression vectors, applied in the field of cellular immunotherapy, can solve the problem of no significant improvement in patient survival

Active Publication Date: 2021-09-07
SHANGHAI LIFE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Gastrointestinal and pancreatic tumors are a great threat to human life. Although surgical treatment, radiotherapy, chemotherapy, and interventional therapy have certain curative effects on such tumors, the survival rate of patients has not been significantly improved.

Method used

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  • Chimeric antigen receptor of cell for targeted expression of Claudin 18.2 and application of chimeric antigen receptor
  • Chimeric antigen receptor of cell for targeted expression of Claudin 18.2 and application of chimeric antigen receptor
  • Chimeric antigen receptor of cell for targeted expression of Claudin 18.2 and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Construction of chimeric antigen receptor expression plasmid containing scFv targeting CLDN 18.2

[0149] By artificially synthesizing a DNA fragment with a length of 1461bp shown in SEQ ID NO.:13, the nucleotides at positions 1-63 encode a leader, and the nucleotides at positions 64-789 encode CLDN 18.2scFv (targeting cancer scFv of embryonic antigen), the nucleotides at 790-924 encode the CD8 hinge region, the nucleotides at 925-996 encode the CD8 transmembrane region, the nucleotides at 997-1122 encode 4-1BB, and the nucleotides at 997-1122 encode 4-1BB. Nucleotides 1123-1458 encode CD3ζ.

[0150] The above DNA fragment was inserted into the downstream of the EF1alpha promoter of the lentiviral expression vector to obtain a chimeric antigen receptor expression plasmid (pLV-CLDN 18.2-scFv-BBz) targeting CLDN18.2. image 3 It is an electrophoresis identification diagram of the restriction endonuclease Xba I restriction endonuclease Xba I inserted into the lentiviral e...

Embodiment 2

[0152] Transfection of T cells with a chimeric antigen receptor expression plasmid targeting CLDN 18.2

[0153] (1) Packaging preparation of lentivirus

[0154] The chimeric antigen receptor expression plasmid, structural plasmid, and envelope plasmid were transfected into 293T / 17 cells at a ratio of 3:2:1 by calcium phosphate transfection method. After 12 hours of transfection, the medium was replaced with fresh DMEM medium containing 10% FBS, and sodium butyrate was added at a final concentration of 5 mM at the same time. After 48 hours of transfection, the cell culture supernatant containing the virus was aspirated into a centrifuge tube, centrifuged at 1500 rpm at 4 °C for 10 min, transferred the supernatant to a new centrifuge tube, filtered through a 0.45 μm filter and stored at -80 °C.

[0155] (2) Preparation of T cells

[0156] Take 10ml of fresh blood from a healthy person, and use lymphocyte separation medium (Yunfei Biology) to separate peripheral blood mononucle...

Embodiment 3

[0161] The specific killing activity of CAR-T cells against CLDN18.2-positive malignant cells

[0162] Take 5×10 4 K562 cells highly expressing CLDN18.2 (CLDN18.2 positive, represented by CLDN18.2-K562) and control K562 cells highly expressing CLDN18.1 (CLDN18.2 negative, represented by CLDN18.1-K562) were inoculated at 96 Orifice plate, add CAR-T cells (CLDN18.2-CART) into the two kinds of cell culture wells according to the three ratios of effector cells (Effector): target cells (Target) = 10:1, 3:1, 1:1 , CAR-T positive control cells (CLDN18.2-CART positive control) and unmodified T cells (T). After incubation for 18 hours, the specific killing activity of CAR-T cells on K562 cells highly expressing CLDN18.2 was detected according to the instructions of the LDH Cytotoxicity Analysis Kit (Beiyuntian). The results showed that the CAR-T cell group (group 1) had a stronger effect on K562 cells highly expressing CLDN18.2 compared with the CAR-T positive control cell group (gro...

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Abstract

The invention discloses a chimeric antigen receptor of a cell for targeted expression of Claudin 18.2 (CLDN 18.2), and particularly discloses a chimeric antigen receptor with an amino acid sequence as shown in SEQ ID NO.14. The chimeric antigen receptor comprises a Claudin 18.2-targeted single-chain antibody, a hinge region, a transmembrane structural domain and an intracellular signal structural domain. The Claudin 18.2-targeted chimeric antigen receptor disclosed by the invention can achieve effective and specific targeted expression of malignant cells (such as tumor cells) of the Claudin 18.2 surface antigen, so that a more efficient method with fewer side effects and adverse reactions is provided for treating some tumors expressing the Claudin 18.2 surface antigen.

Description

technical field [0001] The invention relates to the field of cellular immunotherapy, in particular to a chimeric antigen receptor targeting cells expressing CLDN 18.2 and its application. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment method, which constructs the expression vector of chimeric antigen receptor (CAR) through molecular biology technology, and introduces the expression vector into immune cells isolated from the human body , to express CAR on the cell surface, expand and culture it, and reinfuse it into the human body. Immune cells expressing CAR can specifically recognize and bind target cells, and kill them by releasing specific immune factors. Although the first-generation CAR-T can mediate the killing effect on tumor cells, it does not transduce proliferation signals and induce cytokine production, and the duration of action in vivo is not long, and can only cause transient T cell proliferation. Therefore, anti-tumor The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/3007C07K16/28C07K14/7051C07K14/70517C07K14/70521C07K14/70578C12N15/86C12N5/0636A61K39/001102A61K39/001182A61P35/00C07K2317/622C07K2317/56C07K2317/565C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/86A61K2039/82A61K2039/812A61K2039/828A61K2039/892A61K2039/844A61K2039/852A61K2039/80A61K2039/884A61K2039/836
Inventor 董坚孙亚如杨圣李文荣聂紫
Owner SHANGHAI LIFE SCI & TECH CO LTD
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