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Chimeric antigen receptor lack of ubiquitination and application of chimeric antigen receptor

A chimeric antigen receptor and antigen recognition technology, which is applied to cancer antigen components, vertebrate antigen components, medical preparations containing active ingredients, etc., can solve the problem of limited scale industrial production and clinical practice, low transformation efficiency, Problems such as difficulty in construction

Active Publication Date: 2020-10-27
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this type of design often limits large-scale industrial production and clinical practice due to factors such as high construction difficulty and low conversion efficiency.

Method used

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  • Chimeric antigen receptor lack of ubiquitination and application of chimeric antigen receptor
  • Chimeric antigen receptor lack of ubiquitination and application of chimeric antigen receptor
  • Chimeric antigen receptor lack of ubiquitination and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Embodiment 1: Vector construction of CAR

[0130] The antigen-specific single-chain antibody (scFv) sequences of CD19 and GD2CAR used in the present invention are respectively derived from clinically used FMC63 and 14g2a sequences. The extracellular segment structure of CAR is composed of CD8α signal peptide sequence, myc tag sequence, scFv sequence, and CD8α hinge sequence in series; the transmembrane sequence is the transmembrane sequence of CD8α; Or CD28 intracellular segment sequence in series with human CD3ζ intracellular segment sequence. The above amino acid sequence and the amino acid sequence in which lysine is mutated into arginine in the intracellular segment are converted into base sequences after codon optimization and synthesized by the company (Qinglan Biotech). The base sequences of all CARs in the present invention are finally cloned into the pHR-hEF1α-IRES-EGFP vector by means of Gibson connection (NEB#E2611L), and the part of the CAR used in the fluo...

Embodiment 2

[0131] Example 2: Human primary T cell culture and lentivirus infection

[0132] Human primary T cells were obtained from healthy informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin sulfate (the above reagents were all purchased from Gibco). In order to maintain the proliferation of T cells, 100 U / ml of hIL-2 (Sigma-Aldrich) was added to the culture medium.

[0133] Preparation of lentivirus: 2.2×10 5 Lenti-X 293T cells (TaKaRa #632180) were resuspended in DMEM medium (Gibco #11995-065) containing 10% fetal calf serum and cultured in 6-well cell culture plates (Corning #CLS3516) for 24 hours. Using the liposome transfection system (Mirus#2300), transfect 500ng lentiviral packaging plasmids pCMVdR8.92 (Addgene#8455) and pMD2.G (Addgene#12259) with 500ng lentiviral plasmids to be packaged according to liposome transfection The operating steps of the manual are added to the Len...

Embodiment 3

[0135] Example 3: Flow Cytometry Analysis

[0136] For staining of cell surface markers: antibodies were diluted in FACS buffer (phosphate buffered PBS+2% fetal bovine serum) and incubated with cells for 25 minutes at 4°C in the dark.

[0137] For staining containing intracellular markers: cells were first fixed with 4% paraformaldehyde (Meilunbio #MA0192) at room temperature for 15 minutes, and then permeabilized using pre-cooled methanol on ice for 50 minutes. Staining was then performed by co-incubating with antibody dilutions (same as surface staining) for 60 minutes in the dark at room temperature. The Zombie Violet Fixableviability Kit (Biolegend #423114) was used to distinguish between live and dead cells.

[0138] Flow cytometry data were acquired by a BD LSR Fortessa machine (BD bioscience) and data analysis was performed using FlowJo software (Tree Star). Antibodies used in flow cytometry are listed below.

[0139]

[0140]

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PUM

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Abstract

The invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular structural domain, a transmembrane structural domain and an intracellular structural domainwhich are sequentially connected, the extracellular domain comprises an antigen recognition region; the intracellular structural domain comprises a costimulatory signal conduction region and a CD3[zeta] intracellular region which are sequentially connected to form a costimulatory signal conduction region-CD3[zeta] intracellular region; the costimulatory signal conduction region-CD3[zeta] intracellular region is a polypeptide formed by mutating lysine in a wild type costimulatory signal conduction region-CD3[zeta] intracellular region into arginine. A method is provided for optimization and transformation of CAR-T, all lysine sites of an intracellular segment of the CAR are mutated into arginine, and ubiquitination modification generated after the CAR is stimulated by an antigen is blocked. The strategy is suitable for different CARs and transformation of different intracellular costimulatory domains, and a solution is provided for the problem that the proliferation ability of CAR-T insolid tumors is poor.

Description

technical field [0001] The invention relates to the field of chimeric antigen receptors, in particular to a ubiquitination-deficient chimeric antigen receptor and its application. Background technique [0002] Chimeric antigen receptor is referred to as CAR (chimeric antigen receptor), and T cells equipped with CARs targeting specific tumor antigens are called CAR-T cells. As a rising star of adoptive cell therapy, CAR-T therapy has achieved remarkable results in the treatment of various tumors, especially blood tumors. In 2017, the U.S. FDA successively approved two commercial CAR-T products targeting CD19 antigen for the treatment of malignant leukemia and lymphoma, with remarkable curative effect. However, CAR-T therapy still has many limitations in the treatment of solid tumors, and the failure of CAR-T cells to effectively and continuously expand in patients caused by many factors is considered to be a major manifestation of limiting the anti-tumor activity of CAR-T. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C12N15/86A61P35/00C07K2319/02C07K2319/03C12N2740/15043A61K39/464412A61K39/464471A61K39/4631A61K2239/38C12N5/0636A61K39/4611C12N15/85A61K38/00C12N15/625C12N2510/00C07K16/2803C07K2317/622C07K16/3084C07K14/70517C07K2319/41A61K35/17C07K14/70521C07K14/70578C12N7/00C12N2740/15022C12N2740/15042C12N2740/15062
Inventor 王皞鹏李文涛邱士真魏平
Owner SHANGHAI TECH UNIV
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