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A chimeric gene based on fcγriiia and its use

A chimeric gene, coding gene technology, applied in the field of genetically engineered immune cells based on FcγRIIIa, can solve the problems of lack of versatility and limit the clinical application of CAR technology, and achieve the effect of improving clinical efficacy

Active Publication Date: 2019-09-20
JIANGSU PURECELL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeting tumor-associated antigens (TAAs) in different tissues requires the design of corresponding specific scFvs, and the constructed CAR molecules are limited to specific tumors and are not universal, which limits the clinical application of CAR technology

Method used

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  • A chimeric gene based on fcγriiia and its use
  • A chimeric gene based on fcγriiia and its use
  • A chimeric gene based on fcγriiia and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1F

[0075] Example 1 Identification of Restriction Endonuclease BamH / Sal1 Double Digestion of FcγRⅢa-BB-ζ Lentiviral Expression Vector

[0076] figure 2 It is the part of the target gene released after the chimeric gene FcγRⅢa-BB-ζ is digested with BamH1 and Sal1 on the lentiviral expression vector pLVX-EF1α vector, and the target gene fragment is 1269bp. Swimming lane 1 is the nucleic acid molecular weight standard, and swimming lane 2 is the double digestion product of BamH1 and Sal1.

[0077] The correct plasmid identified by double enzyme digestion was sent to Shanghai Sangon Bioengineering Co., Ltd. to sequence the inserted chimeric gene fragment. The correct plasmid was named pLVX-FcγRⅢa-BB-ζ.

Embodiment 2F

[0078] Example 2 Preparation of FcγRⅢa-BB-ζ lentivirus

[0079] The FcγRIIIa-BB-ζ chimeric gene was designed and constructed as described herein. The lentivirus preparation and purification of the chimeric gene FcγRⅢa-BB-ζ adopts the following method:

[0080] 1. Use the Qiagen EndoFree Plasmid Maxi Kit to extract the plasmid;

[0081] 2. Prepare 293T cells in 100mm culture dish in advance;

[0082] 3. After digesting the cells with trypsin, draw 10ml of complete medium with an electric pipette, pipette all the cells into a single-cell suspension, and transfer them to other 100mm culture dishes in proportion; 37°C, 5% CO 2 incubator overnight;

[0083] 4. Transfect when the cell confluency is 70%-80%;

[0084] 5. Replace the cell medium and replace the whole amount with fresh serum-free DMEM medium;

[0085] 6. Prepare plasmid mixture: take a new 1.5ml centrifuge tube, add 0.5mL DMEM and plasmid;

[0086] 7. Prepare PEI mixture: take a new 1.5ml centrifuge tube, add 0.5m...

Embodiment 3

[0102] Example 3 Preparation of cell product of chimeric FcγRⅢa-BB-ζ gene

[0103] The cells come from patients or healthy donors, and peripheral blood mononuclear cells (PBMC) are obtained by venous blood sampling or blood component apheresis. The method of T cell culture adopts CD3, CD28 monoclonal antibody coating culture flask to activate T cell method, or adopts the paramagnetic polypropylene beads T cell activation method coated with CD3 and CD28 monoclonal antibody, NK cell culture method is carried out as described (Hiroyuki et al., Cancer Res 2009;69:4010-4017). Lentiviral transduction was performed as described (Levine et al., 2006, Proc Natl Acad Sci USA 103:17372-17377).

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Abstract

The invention relates to an Fc gamma RIII a-based chimeric gene and application thereof. The chimeric gene comprises an Fc gamma RIII a signal peptide, an Fc gamma RIII a extracellular area, a CD8alpha cross-film area and an intracellular signal transmission structural domain, wherein the Fc gamma RIII a extracellular area is directly connected with the CD8alpha cross-film area. According to the mosaic gene, the Fc gamma RIII a extracellular area is directly connected with the CD8alpha cross-film area, and a CD8alpha hinge area in a conventional chimeric antigen receptor (CAR) molecule design is deleted, so that an Fc gamma RIII a-CAR molecule is better for activating effector cells, and the killing capability of the Fc gamma RIII a-CAR molecule to tumor cells is remarkably improved; the designed Fc gamma RIII a-CAR molecule combined with a monoclonal antibody drug can be generally used in cell therapy of various tumors.

Description

technical field [0001] The present invention relates to the technical field of tumor biotherapy, in particular to an FcγRIIIa-based chimeric gene and its application, and also to an FcγRIIIa-based genetically engineered immune cell and its application. Background technique [0002] Monoclonal antibodies have gradually become a mainstay in cancer treatment. The mechanism by which monoclonal antibodies play a therapeutic role is mainly to kill target cells through antibody-dependent cell-mediated cytotoxicity (antibody-dependent cellular cytotoxicity, ADCC). In the clinical application of monoclonal antibodies, unsatisfactory therapeutic effects are often observed. The reason is that after radiotherapy and chemotherapy, the effector cells of the monoclonal antibody drug ADCC are exhausted, so that the monoclonal antibody drug cannot fully exert its effect. [0003] Chimeric antigen receptor (CAR) CD19 T cells (CART-19 cells) have achieved remarkable success in the treatment ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/63C12N5/10A61K38/17A61K45/06A61P35/00A61P31/12
CPCA61K35/15A61K35/17A61P31/12A61P35/00C07K14/70535C07K2319/02C07K2319/03C12N15/86C12N2740/15043
Inventor 孙振华
Owner JIANGSU PURECELL BIOMEDICAL TECH CO LTD
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