Engineered immune cells targeting cd47 with a suicide gene switch

A cell-targeted technology, applied in genetic engineering, cells modified by introducing foreign genetic material, plant genetic improvement, etc.

Active Publication Date: 2022-08-02
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no mature CAR-T cell targeting CD47 in this field, and there is an urgent need to develop CAR-T cells targeting CD47 in this field

Method used

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  • Engineered immune cells targeting cd47 with a suicide gene switch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Example 1 Isolation of PBMC and expansion of T cells from donor blood

[0205] Monocytes were isolated from umbilical cord blood, subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich), and enriched for T cells (EasySep human T cell enrichment kit, Stemcell Technologies) using conjugated anti-CD3 / anti-CD28 T cells were activated and expanded by magnetic beads; the medium was X-vivo15 (5% FBS, 2mM L-glutamine, 1mM sodium pyruvate, 300IU / ml rhIL2); all cells were placed at 37°C, 5% CO 2 Cultured in a constant temperature incubator.

Embodiment 2

[0206] Example 2 Cell Culture

[0207] Human ovarian cancer tumor cell lines A1847 and SKOV-3; pancreatic cancer cells BxPC3 and PANC-1; BxPC3-ffluc cell lines were screened by using fireflyluciferase lentivirus to infect BxPC3 cells, and the above cells were cultured in RPMI1640 medium; 293T (ATCC -CRL3216) was cultured in DMEM medium. All media were supplemented with 10% (v / v) fetal bovine serum and 100 U / ml of penicillin and streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate.

Embodiment 3

[0208] Example 3 CAR structure design and transduction

[0209] Design and construct CAR-T cells targeting CD47, wherein the CAR-T cells stably express the CAR gene. CAR is an artificially designed amino acid sequence, including a signal peptide, scFv, hinge region, transmembrane region and intracellular signal region connected in sequence.

[0210] In addition, the CAR-T cells targeting CD47 constructed in this example stably express the iCasp9 gene. iCasp9 gene is an artificially designed amino acid sequence, including human FK506 binding protein F36V mutant (FKBP-F36V), human caspase 9 protein (CASP9, GenBank number, NM001229), in which the endogenous apoptosis and recruitment domains of human caspase 9 protein are deleted , FKBP-F36V and CASP9 are linked by Ser-Gly-Gly-Gly-Ser to form FKBP-F36V-SGGGS-CASP9.

[0211] The vector for CAR and iCasp9 gene expression can be DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon or other gene tra...

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Abstract

The present invention provides an engineered immune cell targeting CD47 with a suicide gene switch. Specifically, the present invention provides a chimeric antigen receptor T cell targeting CD47, and the cell suicide element is included in the CAR structure of the cell. The present invention optimizes the affinity of the CD47 scFv in the CAR structure and selects a suitable CAR-T cell infusion method, thereby reducing the toxicity to normal tissues to an acceptable level for humans. At the same time, on the basis of ensuring the activity of CD47 CAR-T cells, cell suicide elements are added to control the activity of CAR-T cells and related toxicity such as CRS to enhance safety.

Description

technical field [0001] The invention relates to the field of immunotherapy, in particular to an engineered immune cell targeting CD47 with a suicide gene switch, and more particularly to a CD47-targeting chimeric antigen receptor T cell and a preparation method and application thereof. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with remarkable curative effect, and a novel treatment method for autoimmune anti-cancer. It is a method of using biotechnology and biological preparations to culture and expand immune cells collected from patients in vitro and then infuse them into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. [0003] Chimeric antigen receptors (CARs) are composed of extracellular antigen recognition region, usually scFv (single-chain variable fragment), transmembrane region and intracellular costimulatory signal region. The extracellular seg...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61P35/00
Inventor曹卫何佳平刘丽萍马安云
OwnerGRACELL BIOTECH SHANGHAI CO LTD