Engineering immune cell with suicide gene switch of targeting human mesothelin

An immune cell and suicide gene technology, applied in genetic engineering, blood/immune system cells, targeting specific cell fusion, etc.

Active Publication Date: 2019-04-09
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, immunotherapeutic methods such as antibodies targeting mesothelin and chimeric antigen receptor T cells (CART) targeting mesothelin have been reported, but it was found that CART constructed with mouse anti-human mesothelin antibody was Toxic side effects such as

Method used

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  • Engineering immune cell with suicide gene switch of targeting human mesothelin
  • Engineering immune cell with suicide gene switch of targeting human mesothelin
  • Engineering immune cell with suicide gene switch of targeting human mesothelin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0206] Isolation of PBMCs and Expansion of T Cells from Donor Blood

[0207] Mononuclear cells were isolated from umbilical cord blood, density gradient centrifugation was performed using Histopaque-1077 (Sigma-Aldrich), and T cells were enriched (EasySep human T cell enrichment kit, Stemcell Technologies), using conjugated anti-CD3 / anti-CD28 T cells were activated, cultured and expanded with magnetic beads; medium used was X-vivo15 (containing 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

[0209] Cell Culture and Construction

[0210] Cell lines expressing MSLN: OVCAR3 cells (human ovarian cancer cell line, HTB-161 TM ), HCT116 (human colon cancer cell line, CCL-247 TM ), CRL5826 (human lung cancer cells, CRL-5826, H226); K562 cells expressing MSLN / CD19 (human erythroleukemia cell line, -CCL-243), the above cells were cultured using RPMI 1640 medium; 293T (human kidney epithelial cell line cells, CRL-3216) were cultured using DMEM medium. All media were supplemented with 10% (v / v) fetal bovine serum and 100 U / ml penicillin and streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate

[0211] Among them, K562 cells expressing MSLN and CD19, and CRL5826, HCT116 and OVCAR3 expressing PDL-1 highly are stably transfected cell lines obtained by transferring MSLN, CD19 and PDL-1 antigens through lentiviral vectors, and then undergoing monoclonal screening. , can specifically express protein molecules such as MSLN, CD19 and PDL-1.

Embodiment 3

[0213] CAR structure design and transduction

[0214] meso-CAR structure, that is, the CAR structure targeting Mesothelin:

[0215] The method of the present invention constructs the first-generation, second-generation and third-generation CARs, all of which have an adjustable switch icasp9 (FKBP12-F36V-Caspase9) in their structures, connected through P2A, such as figure 1 shown.

[0216] The core structure of CAR includes the CD8 extracellular leader sequence, the scFv from P4 (specifically targeting mesothelin), the hinge from CD8 and the CD8 / CD28 transmembrane region. According to the presence or absence of co-stimulatory signals in intracellular segments, four meso-CARs were constructed, named CD3ζ, 4-1BB-CD3ζ, CD28-CD3ζ, and CD28-4-1BB-CD3. Named according to the different intracellular co-stimulatory regions, as shown in Table 1:

[0217] Table 1 Name of CAR-T cells

[0218] name

co-stimulatory signal

intracellular activation domain

P4-z-CAR (...

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Abstract

The invention provides an engineering immune cell with a suicide gene switch of targeting human mesothelin (MSLN). Particularly, the invention provides a chimeric antigen receptor T cell of the targeting human mesothelin, a CAR (Chimeric Antigen Receptor) structure of the cell comprises a cell suicide element, and the expression of a PD1 gene in the cell is silent. Particularly, the CAR structurecomprises a CAR basic structure and the cell suicide element at the same time, the CAR basic structure and the cell suicide element are independent of each other, and the corresponding functions of the CAR basic structure and the cell suicide element are non-interfering. In addition, the silent expression of the PD1 gene in the cell has a synergistic effect with the CAR structure, the tumor killing effect is strengthened, and the disease does not relapse easily.

Description

technical field [0001] The invention relates to the field of immune cell therapy, in particular to an engineered immune cell targeting human mesothelin with a suicide gene switch. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back 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 immune antigen receptors (Chimeric antigen receptors, CARs) are composed of extracellular antigen recognition region, usually scFv (single-chain variable fragment), transmembrane region and intracellular co-stimulatory signal region. The extracellular segment of CARs can recognize a specific antigen, and then transduce the ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K19/00A61P35/00
CPCA61K35/17C12N5/0636C12N5/0646C07K14/7051C07K16/28C07K2319/02C07K2319/03C07K2319/33C07K2317/622C07K2319/74C12N2510/00A61P35/00C07K19/00C12N5/10C12N15/867
Inventor 曹卫刘丽萍何佳平马安云
Owner GRACELL BIOTECH SHANGHAI CO LTD
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