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Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and application of bispecific chimeric antigen receptor

A chimeric antigen receptor and bispecific technology, applied in the field of tumor biological immunotherapy, can solve the problems of weak targeting specificity, limited proliferation and killing ability, and low anti-tumor efficiency, so as to improve prognosis and increase specificity. Sexually lethal effects

Pending Publication Date: 2020-11-27
金鑫
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to overcome the above-mentioned problems faced in the biological immunotherapy of tumors (that is, the immune response cells in the tumor microenvironment, including T cells and NK cells, recognize and kill tumors, the targeting specificity is not strong and subject to various inhibitions, proliferation and limited killing ability and thus low anti-tumor efficiency), a bispecific chimeric antigen receptor amino acid construct or functional variant thereof targeting CD123 and NKG2DL at the same time, its encoding nucleotide sequence, and its expression Vector, engineered bispecific chimeric antigen receptor-modified immune response cells targeting CD123 and NKG2DL and applications thereof

Method used

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  • Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and application of bispecific chimeric antigen receptor
  • Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and application of bispecific chimeric antigen receptor
  • Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and application of bispecific chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Construction of an expression plasmid targeting bispecific chimeric antigen receptors targeting human CD123 and human NKG2DL

[0127]This example provides a bispecific chimeric antigen receptor that simultaneously expresses anti-human CD123 scFv (CSL362-humanized mouse anti-CD123 mAb 7G3) and the extracellular domain of human NKG2D. In this example, the two antigen-binding domains are combined in parallel. Such as figure 1 As shown, the structure of the bispecific chimeric antigen receptor provided in this example includes: the first signal peptide domain (human CD8α, CD8αSP) connected in sequence, the first antigen binding domain (anti-human CD123 targeting human CD123 scFv, Anti-CD123 scFv), the first hinge domain (human CD8α hinge region, CD8αH), the first transmembrane domain (human CD28 transmembrane region, CD28 TM), the first co-stimulatory signal domain (human CD28 Stimulatory signal domain, CD28 CS), the first intracellular signaling domain (human C...

Embodiment 2

[0141] The preparation of embodiment 2 lentiviruses

[0142] 1. Virus packaging: The expression plasmid of the bispecific chimeric antigen receptor targeting human CD123 and human NKG2DL constructed in Example 1, and the viral packaging plasmid (pLP1, pLP2, VSVG) were 7:5:5: The ratio of 3 (total 24ug) was co-incubated with Lipofilter (Hanheng Company) and then co-transfected into 293T cells (100mm culture dish, confluence 50%-70%). For specific transfection procedures, please refer to the instructions of Hanbio LipoFiter Transfection Reagent. The DMEM complete medium was replaced 6 h after transfection.

[0143] 2. Virus harvest and concentration: Collect the cell culture supernatant at 48 hours and 72 hours respectively, centrifuge at 3000 rpm at 4°C for 15 minutes, and filter the supernatant through a 0.45 μm filter membrane. Finally, ultracentrifuge at 25,000 rpm for 2 to 3 hours at 4° C., discard the supernatant, and resuspend the pellet with fresh DMEM medium to obtain...

Embodiment 3

[0144] Example 3 Preparation of bispecific chimeric antigen receptor T cells (named 123NL-CART) targeting human CD123 and human NKG2DL

[0145] Step 1 Obtain human peripheral blood T cells:

[0146] 1. Collect about 20ml of human peripheral fresh blood with a vacuum blood collection tube containing heparin;

[0147] 2. Take a 50ml centrifuge tube, draw 20ml of Ficoll lymphocyte separation solution into the centrifuge tube, tilt the tube, and slowly add the collected peripheral blood to the upper layer of Ficoll along the tube wall at about 1cm above the Ficoll liquid level. Centrifuge at 500g for 25min at 4°C.

[0148] 3. After centrifugation, it is divided into four layers from the bottom of the tube to the liquid surface, which are red blood cell and granulocyte layer, stratified liquid layer, mononuclear cell layer (ie, buffy coat layer), and plasma layer. Use a pipette to directly suck up the upper layer of plasma, gently suck out the mononuclear cell layer, put it into ...

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Abstract

The invention discloses a bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and the application of the bispecific chimeric antigen receptor, and particularly discloses a bispecific chimeric antigen receptor (CAR) amino acid construct or a functional variant thereof, wherein the bispecific chimeric antigen receptor (CAR) amino acid construct can simultaneously expresses an anti-CD123 single-chain antibody and a natural NKG2D extracellular fragment and can target CD123 and NKG2DL; the bispecific chimeric antigen receptor has a parallel connection mode and a series connectionmode; and the invention discloses a CAR (chimeric antigen receptor), a construct structure thereof, a nucleotide sequence of the construct, a recombinant expression vector containing the nucleotide sequence and a construction mode and application of the corresponding expression vector. The CAR structure endows T cells with higher and lasting multiplication capacity and high anti-tumor capacity; and according to the bispecific chimeric antigen receptor, multiple infusions of single-target CAR-T cells can be avoided, so that not only is the harm to a patient reduced, but also the economic pressure of the patient can be reduced, and the bispecific chimeric antigen receptor has relatively great clinical research and application values.

Description

technical field [0001] The invention relates to the field of tumor biological immunotherapy, and relates to a bispecific chimeric antigen receptor (CAR). Specifically, it relates to a bispecific chimeric antigen receptor (CAR) targeting human CD123 and human NKG2DL simultaneously, comprising anti-human CD123 scFv and human NKG2D extracellular domain or functional variants thereof, a recombinant expression vector, the CAR-modified immune response cells, and preparation methods and applications thereof. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) therapy is to artificially express an antigen-binding domain that can recognize specific tumor surface antigens on the surface of T cells through genetic engineering technology, so that T cells can obtain specific recognition of antibodies The ability of tumors, and activate T cells through the intracellular signaling region of CAR, so that CAR-T cells release cytokines, proliferate and exert cytotoxicity, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/113C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00A61P35/02C12R1/93
CPCC07K16/2866C07K14/7056C07K14/7051C12N5/0636C12N15/86C12N7/00A61K39/0011A61P35/00A61P35/02C07K2317/622C07K2319/32C07K2319/33C07K2319/02C07K2319/03C12N2740/15021C12N2740/15043C12N2510/00C12N2800/107A61K2039/5158
Inventor 赵明峰金鑫曹雅青
Owner 金鑫
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