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Chimeric antigen receptor mechanocyte as well as building method and application thereof

A technology of chimeric antigen receptors and fibroblasts, applied in the fields of medicine, biomedicine, and genetic engineering

Active Publication Date: 2018-11-13
ZHEJIANG K2 ONCOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the prior art, there are no relevant research reports on the use of chimeric antigen receptors (CAR) to engineer fibroblasts

Method used

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  • Chimeric antigen receptor mechanocyte as well as building method and application thereof
  • Chimeric antigen receptor mechanocyte as well as building method and application thereof
  • Chimeric antigen receptor mechanocyte as well as building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction of a plasmid comprising a chimeric antigen receptor protein of an anti-human EpCAM single-chain antibody

[0069] The chimeric antigen receptor protein comprising the single-chain antibody of anti-human epithelial cell adhesion molecule (EpCAM) comprises from N-terminal to C-terminal sequence: 1) signal peptide sequence; 2) anti-human EpCAM single-chain antibody sequence; 3) including trans The junction sequence of membrane sequence; 4) mouse CD8 intracellular sequence or GFP sequence; 5) HAtag sequence ( figure 1 ). The sequence information involved is detailed in the table below:

[0070]

[0071]

[0072] The nucleotide and amino acid sequences of the signal peptide are shown in SEQ ID NO.1 and 2; the encoded nucleotide and amino acid sequences of the anti-human EpCAM single-chain antibody are shown in SEQ ID NO.11-22; including the transmembrane sequence The nucleotide and amino acid sequences of the connecting sequence are shown in ...

Embodiment 2

[0075] Example 2: Lentivirus packaging, concentration and titer determination

[0076] 1. Experimental materials:

[0077] 1) Virus packaging plasmids: pCMVΔR.89 plasmid (Pcg41), pVSV-Gplasmid plasmid (Pcg10).

[0078] 2) Packaging cells: Hek-293T cells (DuBridge, et. al. 1987, Pear, et al. 1993).

[0079] 3) Virus packaging medium: DMEM complete medium (Dulbecco's Modified Eagle Medium, Invitrogen, catalog #11995); fetal bovine serum (FBS, Hyclone, catalog #: SH30084.03); non-essential amino acids (Gibcocatalog #1140-050); Antibiotics (PenStrep, Gibic, Cat. No. 15070063).

[0080] 4) Cell transfection reagent: Lipofectamine2000 (Invitrogene, product number: 11668-019); serum-free medium Opti-MEM (Gibco, catalog #11058);

[0081] 5) Virus purification and infection: 0.45 μM filter (Corning, catalog #430314); sucrose (Sigma, catalog #: S7903); polybrene (Polybrene, Sigma, catalog #: H9268).

[0082] 6) Detection antibody: HAtag antibody (GenScript, catalog number: A01244). ...

Embodiment 3

[0094] Example 3: Infection of Target Cells and Screening of Chimeric Antigen Receptor Stable Expression Cell Lines

[0095] 1. Mouse embryonic fibroblast (MEF) preparation

[0096] 1) Sexually mature female mice were caged with male mice, and the vaginal opening of the mice was observed to have jelly-like substances. The morning was defined as 0.5 days of pregnancy.

[0097] 2) Execute the female mouse at 12.5-14.5 days pregnant, take out the embryos with fetal membranes, place them on a sterile plate, wash with PBS and discard the red blood cells. The fetal membranes were peeled off, the fetal mice were taken out, and washed three times with PBS. Remove the head, viscera and limbs of the embryo, place the trunk in a sterile plate, wash with PBS at least three times, and discard the red blood cells. Cut mouse embryo torso to 1mm 3 The following fragments are sucked into the centrifuge tube.

[0098] 3) Add an appropriate amount of trypsin, incubate at 37°C for 10-20 minut...

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Abstract

The invention provides an engineering transformation mechanocyte as well as a building method and application thereof. The engineering transformation mechanocyte is primary mechanocyte built through separation from human or mice, or is a mechanocyte or cell line for expressing chimeric antigen receptor protein at the cytomembrane surface through cell engineering transformation on the basis of cellline; a specific target resistant single-chain antibody is expressed at the extracellular region of chimeric antigen receptor protein. The engineering transformation mechanocyte is named as ''CAR-F''; the CAR-F can be combined with tumor epithelial cells and is applied to tumor disease treatment or in vitro detection.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering, medicine and biomedicine, and in particular relates to an engineered chimeric antigen receptor fibroblast, a pharmaceutical composition and a kit containing the chimeric antigen receptor fibroblast, and a chimeric antigen receptor fibroblast. The application of chimeric antigen receptor fibroblasts and chimeric antigen receptor protein in the diagnosis and treatment of tumor diseases. Background technique [0002] Fibroblasts, also known as fibroblasts, are the most common cells in connective tissue, which are differentiated from mesenchymal cells in the embryonic period. Fibroblasts have large cell bodies, multi-projected spindle-shaped or star-shaped flat cells, regular oval nuclei, and indistinct cell outlines. In connective tissue, fibroblasts also exist in the form of fibroblasts in their mature state, and the two can transform into each other under certain conditions. Differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C07K16/30C12N15/62C12N7/01A61K35/17A61K39/395A61P35/00
CPCA61K35/17A61K39/39558A61P35/00C07K16/30C07K2317/622C07K2319/02C07K2319/03C12N5/0656C12N7/00C12N2510/02
Inventor 孙志坚康平
Owner ZHEJIANG K2 ONCOLOGY CO LTD
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