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CD22-targeted high-lethality chimeric antigen receptor T-cell and use thereof in preparation of drugs for treating tumors

A chimeric antigen receptor and cell technology, applied in the field of medicine and biology, can solve the problem of low killing ability of tumor cells

Active Publication Date: 2016-09-28
深圳生创精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the defect that the existing T cells expressing anti-CD22 chimeric antigen receptors have relatively low killing ability to kill CD22 positive tumor cells, and provide a T cell expressing anti-CD22 chimeric antigen receptors with high killing ability. somatic T cells

Method used

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  • CD22-targeted high-lethality chimeric antigen receptor T-cell and use thereof in preparation of drugs for treating tumors
  • CD22-targeted high-lethality chimeric antigen receptor T-cell and use thereof in preparation of drugs for treating tumors
  • CD22-targeted high-lethality chimeric antigen receptor T-cell and use thereof in preparation of drugs for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Synthesize the DNA shown in SEQ ID NO.3 by using the full sequence synthesis method. In SEQ ID NO.3, the 1st-6th position is the upstream enzyme-cut connection sequence (the endonuclease is EcoRI), and the 7th-66th position is Signal peptide coding sequence, the 67th-1467th position is the coding sequence (as shown in SEQ ID NO.2, encoding the polypeptide shown in SEQ ID NO.1), and the 1468-1473rd position is the downstream enzyme cutting linking sequence (endonuclease for MluI).

Embodiment 2

[0037] The pLVX-IRES-ZsGreen1 purchased from Clontech Company with the product number of 631982 was used as a vector, and the DNA coding sequence shown in SEQ ID NO.2 in Example 1 was inserted into it. Specifically, pLVX-IRES-ZsGreen1 and the DNA shown in SEQ ID NO.3 synthesized in Example 1 were digested by endonuclease EcoRI and endonuclease MluI respectively, and ligated. After the linker was transformed and verified to be correct , to obtain the lentiviral expression vector pLVX-IRES-CAR of this embodiment.

Embodiment 3

[0041] The lentiviral expression vector obtained in Example 2 and Comparative Example 2 and the pLVX-IRES-ZsGreen1 empty vector were respectively packaged for lentivirus, and then T cells were cultured, infected and expanded in vitro according to the following methods.

[0042] Isolate mononuclear cells in blood according to the following method: mix 1mL sterile PBS with 1mL; then gently add the upper layer of Ficoll; centrifuge at 400×g at 4°C for 30min, set the acceleration and deceleration to 0; gently remove the upper layer Add PBS to resuspend and wash the cells; centrifuge at 100×g for 10 minutes, with normal acceleration and deceleration, remove the upper washing solution after centrifugation; culture with 1mL1640+10%FBS+1% double antibody+1×Glutamine Base resuspended cells. Then use anti-human CD3 / CD28 magnetic beads (purchased from Thermo Fisher) to stimulate amplification, concentration 1×10 6 Cells / mL, add 100 μL magnetic beads, then add 30IU / mL rhIL-2 (CellGenix),...

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Abstract

The invention provides an anti-CD22 chimeric antigen receptor. An amino acid sequence of the anti-CD22 chimeric antigen receptor comprises a sequence shown in SEQ ID No. 1; the anti-CD22 chimeric antigen receptor can be used for identifying CD22 protein and can be used for activating tyrosine signal channels after identifying the CD22 protein. The invention further provides a coding nucleic acid of the anti-CD22 chimeric antigen receptor, an expression vector, a cell, a pharmaceutical composition and use of the coding nucleic acid, the expression vector, the cell and the pharmaceutical composition. By the technical scheme, a T lymphocyte expressing the anti-CD22 chimeric antigen receptor has high-specificity cell toxic effect and very-high killing capability on tumor cells expressing CD22.

Description

technical field [0001] The present invention relates to the field of medical biotechnology, in particular to a CD22-targeted chimeric antigen receptor, its encoding nucleic acid, expression vector, cell, pharmaceutical composition and applications thereof. Background technique [0002] Chimeric antigen receptor (CAR) is an artificially synthesized T cell receptor, which consists of an extracellular targeting linker region (single-chain antibody or ligand) and a T cell activation signal domain (hinge region, Transmembrane region, intracellular signal transduction region). The extracellular targeting linking region is composed of a single-chain antibody or a ligand, and has the function of specifically binding the target antigen. The transmembrane region has the function of transmitting signals, and the intracellular signal region adopts the intracellular signal region of immunoreceptor tyrosine activation motif (ITAM) CD3ζ or FCSRIY and co-stimulatory signal molecules CD28, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/70596C07K16/2803C07K2319/02C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 赵琦刘遥项春生李程
Owner 深圳生创精准医疗科技有限公司
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