System for inhibiting pathological target cells in space-time adjustable manner

A target cell, pathological technology, applied in the field of tumor immunology, can solve the problem that the toxic effect cannot be ignored

Active Publication Date: 2015-12-30
CARSGEN THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its toxic effect cannot be ignored

Method used

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  • System for inhibiting pathological target cells in space-time adjustable manner
  • System for inhibiting pathological target cells in space-time adjustable manner
  • System for inhibiting pathological target cells in space-time adjustable manner

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Experimental program
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preparation example Construction

[0060] Conventional methods for preparing "chimeric antigen receptor immune effector cells" are known to those skilled in the art, including allowing them to express the intracellular domains of intracellular co-stimulatory cell molecules, such as CD28 (preferably including CD28a, CD28b) , CD137, CD27, CD3ζ (preferably CD3ζ intracellular domain), CD8, CD19, CD134, CD20, one or more of FcRγ. Through their combination with corresponding ligands, the second signal of immune effector cells is activated, the proliferation ability of immune cells and the secretion function of cytokines are enhanced, and the survival time of activated immune cells is prolonged.

[0061] In the present invention, the pathological target cells may be various harmful cells in the body that are not conducive to health and must be removed from the body. The pathological target cells include tumor cells. Any tumor known in the art can be included in the present invention, as long as the tumor can express ...

Embodiment 1

[0071] Example 1. Construction of anti-human EGFRVIIIWTE-CH12 antibody recombinant plasmid in the present invention

[0072] 1. Amplification of nucleic acid fragments

[0073] (1) Using the antibody pH / CH12 as a template (see SEQ ID NO: 36 for the sequence). The upstream primer 5'-gatgtgcagcttcaggagtcggg-3' (SEQ ID NO: 1) and the downstream primer 5'-acaataatatgtggctgtgtcc-3' (SEQ ID NO: 2) PCR amplified the CH12VH fragment, and the PCR amplification conditions were pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 58°C, 40s; extension: 68°C, 40s; 27 cycles, and then a total extension of 68°C, 10min. The size of the amplified product was 288bp, which was in line with the expectation.

[0074] (2) Amplification of the heavy chain signal peptide-WTE fragment, the primers are as follows:

[0075] 5'-cctagctagccaccatgagagtgctgattcttttgtggctgttcacagcctttcct-3' (SEQ ID NO: 3),

[0076] 5'-agctgtggagccagacaggaaaccaggaaaggctgtgaacagccac-3' (SEQ ID NO: 4),

[0077] ...

Embodiment 2

[0101] Example 2, Expression and purification of anti-human EGFRvIIIWTE-CH12 antibody

[0102] 1. Expression of anti-human EGFRvIIIWTE-CH12 antibody

[0103] Free-Style293-F cells (purchased from Invitrogen) were used for antibody expression, and the suspension culture and transfection methods were in accordance with FreeStyle TM 293ExpressionSystem manual operation. Specifically, the cell density was adjusted to 1×10 before transfection. 6 Cells / mL, blow off and make the cells free of clumps, and use trypan blue staining to determine the cell viability > 95%. Transfection step: Dilute 52μgpH / WTE-CH12H and 48μgpK / WTE-CH12K (molar ratio 1:1) recombinant plasmids and 200μL Free-Style293-F cell liposome transfection reagent "293fectin" to 3.33mL with Opti-MEM After standing still for 5 minutes, the plasmid and transfection reagent were slowly mixed, and reacted at room temperature for 20 minutes to form a DNA-fectin mixture, and then added 93.3mL Free-Style293-F cells (density...

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Abstract

The invention relates to a system for inhibiting pathological target cells in a space-time adjustable manner, and discloses a technical scheme based on the tumor specificity chimeric antigen receptor (CAR) technology. The CAR immunologic effector cells can target the pathological target cells only under a condition that a mediate material exists, so as to realize continuous multiplication and play the killing effect on tumor cells; under the condition that the mediate material does not exist, the CAR immunologic effector cells do not play the function. According to the system, a solution is provided for avoiding in-vivo continuous multiplication of the CAR immunologic effector cells and the toxic effect generated on normal tissue cross reaction of the CAR immunologic effector cells.

Description

technical field [0001] The present invention relates to the field of tumor immunology, and more specifically, relates to a system for inhibiting pathological target cells with space-time regulation. Background technique [0002] With the development of tumor immunology theory and technology, the role of immunotherapy in tumor treatment has been paid more and more attention. T lymphocytes play a major role in the tumor immune response. In recent years, the immune effector cells that express tumor-specific chimeric antigen receptors (Chimericantigenreceptors, CARs) using genetic modification technology have shown high targeting, killing activity and persistence. , injecting a new solution for adoptive cellular immunotherapy. CAR is a single-chain antibody (scFv) or antibody fragment that recognizes a tumor-associated antigen (TAA) and an activation sequence of a T cell or NK cell that are genetically recombined in vitro to form a recombinant plasmid that is transfected and pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K47/42A61P35/00C07K14/71C07K16/30
CPCA61K38/00C07K14/70517C07K2319/00C07K16/18C07K16/44C07K2319/33C07K2319/40A61P35/00A61K35/17C07K14/7051C07K14/70521C07K14/70578C07K16/2863C07K2317/622C07K2319/02C07K2319/03A61K39/00A61K2039/505
Inventor 王华茂
Owner CARSGEN THERAPEUTICS
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