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Manually target-modified CAR-T cells and preparation method and application thereof

A technology of cells and modifiers, applied in the field of artificially modified CAR-T cells and its preparation, can solve the toxic and side effects of the body, can not solve the problems of tumor off-target effects, cell activity reduction, etc., to achieve the protection of functional activity, Overcoming off-target phenomena and tumor immune escape, avoiding effects

Active Publication Date: 2019-08-23
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, direct chemical targeting of CAR-T cells often results in reduced cell viability and impaired motility and migration.
At this stage, the main problem of these CAR-T cell engineering technologies is to reduce the biological function activity of cells, which cannot solve the problem of tumor off-target effects well, and also has certain potential toxic side effects on the body
In view of these technical bottlenecks, it is urgent to develop a safe and effective CAR-T tumor-targeted therapy technology to overcome the off-target phenomenon and tumor immunosuppression in the process of cell therapy.

Method used

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  • Manually target-modified CAR-T cells and preparation method and application thereof
  • Manually target-modified CAR-T cells and preparation method and application thereof
  • Manually target-modified CAR-T cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of sugar / lipid derivatives modified by chemical reporter groups

[0049] Synthesis of carbohydrate derivatives: We first synthesized 4-nitrophenyl chloroformate-activated bicyclic [6.1.0 ] Nonene (nPC-BCN). The nPC-BCN was then coupled to a glycan (mannose) to give N-BCN-carbonyl-D-mannosamine (ManN-BCN). To increase cellular uptake, these unnatural glycans were peracetylated to form tetra-O-acetyl compounds and lyophilized to obtain tetraacetylated-N-BCN-carbonyl-D-mannosamine (Ac 4 ManNBCN) powder.

[0050] Synthesis of lipid derivatives: mainly refer to the method disclosed in the literature Analytical Chemistry 2013 85 (10), 5263-5270. The specific preparation method is as follows: dissolve a certain amount of 1,2-dibromoethane and sodium azide in DMF, react at 80°C for 20 hours, add ice water and sodium chloride solution after the reaction, and extract to obtain the intermediate 2' - bromoazidoethane. Dissolve the obtained 2'-bromoazid...

Embodiment 2

[0051] Example 2: Artificial bioorthogonal targeted CAR-T cells for the treatment of hematological tumors

[0052] Azide group modification of CAR-T cells: CAR-T cells were co-incubated with azide-modified choline derivatives (AE-Cho) at a final concentration of 32-64 μM at 37°C for 48 h, and the lipids of T cells Metabolic process, the azide group is embedded in the surface of the CAR-T cell membrane (N 3 -CAR-T). The excess modifier was washed away with PBS buffer, and the cells were stained with DBCO-Fluor 488. Cells were harvested and analyzed by confocal and flow cytometry for the expression of azide molecules on the cell surface. Such as image 3 As shown, azide molecules are highly expressed on the surface of CAR-T cells.

[0053] Glucose metabolism modification of hematoma cells (Raji): Luci-Raji cells carrying luciferase were mixed with BCN group-modified mannose (Ac 4 ManNBCN) were co-incubated at 37°C for 48 hours, and the -BCN group was embedded on the tumor...

Embodiment 3

[0056] Example 3: Artificial bioorthogonal targeted CAR-T cells for the treatment of solid tumors

[0057] Azide group modification of CAR-T cells: CAR-T cells were co-incubated with azide-modified choline derivatives (AE-Cho) at a final concentration of 64 μM at 37°C for 48 h, through the lipid metabolism of T cells process, the azide group is embedded in the surface of the CAR-T cell membrane (N 3 -CAR-T). The excess modifier was washed away with PBS buffer, and the cells were stained with DBCO-Fluor 488. Cells were harvested and analyzed by confocal imaging and flow cytometry for the expression of azide molecules on the cell surface. Such as image 3 As shown, azide molecules are highly expressed on the surface of CAR-T cells.

[0058] Glycometabolic Modification of Raji Solid Tumors: Divide 1 × 10 7 Luci-Raji cells carrying luciferase subcutaneously in tumor-bearing NOD / SCID mice. When the tumor volume reaches 100mm 3 Around time, by intratumoral injection of 5-10...

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Abstract

The invention relates to the technical field of biological engineering, in particular to metabolically modified CAR-T cells and a preparation method and application thereof. According to the method, through biological orthogonal metabolic modification, the CAR-T cells can be effectively targeted and drawn to a tumor site in vivo; in-vivo targeting and drawing leads to combination of specific CAR molecules on the CAR-T cells with tumor antigens on the tumor site, correspondingly the specific activation of the CAR-T cells is stimulated, and a more efficient tumor killing capability is achieved.

Description

technical field [0001] The present invention relates to the technical field of bioengineering, in particular to CAR-T cells modified by artificial metabolism and its preparation method and application. Background technique [0002] Adoptive cell therapy (ACT) is an effective anticancer strategy. T cells engineered to express chimeric antigen receptors (Chimeric Antigen Receptors, CARs), known as "living drugs", have shown remarkable efficacy in cancer immunotherapy, especially in the treatment of blood cancers, and have achieved great success. However, as a monotherapy, CAR-T cell immunotherapy still faces great challenges in terms of efficacy and safety. Toxicity caused by "off-target effects" and resulting dysplasia of normal B cells is an important potential side effect of CAR-T cell therapy. Another major obstacle is that tumor immune escape has become a major obstacle to the success of CAR-T cells in the treatment of solid malignancies. Tumor cells alter or hide tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K2319/00C12N5/0636C12N2510/00
Inventor 蔡林涛潘宏马轶凡李文军罗英梅王芳芳
Owner SHENZHEN INST OF ADVANCED TECH
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