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Chimeric antigen receptor for high-efficiency orientation amplification in vitro and application thereof

A chimeric antigen receptor and carrier technology, applied in the fields of genetic engineering and immune cell therapy, can solve problems such as limited progress

Active Publication Date: 2018-05-11
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although different research teams have improved the infection efficiency of T cells to CAR-encoded viruses through electroporation, increased virus MOI, and secondary infection, and increased the proportion of CAR-positive cells to a certain extent, the progress made is very limited.

Method used

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  • Chimeric antigen receptor for high-efficiency orientation amplification in vitro and application thereof
  • Chimeric antigen receptor for high-efficiency orientation amplification in vitro and application thereof
  • Chimeric antigen receptor for high-efficiency orientation amplification in vitro and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0190] Example 1. Preparation of specific and selective CAR molecules

[0191] Specific primers were designed, and the leader peptide domain (SEQ ID No.1), the coding sequence of the hinge region and the transmembrane region (SEQ ID No. 2) Amplify the coding sequence (SEQ ID No.3) of the intracellular signal transduction domain CD3ζ of the T cell receptor protein CD3 molecule and the intracellular signal activation domain of the CD137 molecule. The specific selectivity domain in the CAR molecule is derived from the 95th-104th amino acid coding sequence (SEQ ID No.4) of the C-terminal domain of human nucleoprotein La / SS-B, which is prepared by chemical synthesis and inserted into Between the VL and VH domains of the humanized single-chain antibody ScFv coding region in the CAR molecule. The structure of the CAR is F 0 -F 1 -L 1 -Z-L 2 -F 2 -H-TM-C-CD3ζ, the amino acid sequence is shown in SEQ ID NO.:8. The above-mentioned different coding sequences were spliced ​​and amp...

Embodiment 2

[0192] Example 2. Construction of Chimeric Antigen Receptor Expression Vector

[0193] The CAR coding sequence (SEQ ID NO.: 7) in Example 1 was cloned into the lentiviral expression vector pLenti-CMV using molecular cloning technology. In order to compare the advantages of the modified CAR molecule in the present invention in preparing chimeric antigen receptor T cells, a conventional CAR molecule targeting CD19 antigen (amino acid sequence shown in SEQ ID NO.: 9, patent No. : CN103492406A) as a control, a CD19-CAR virus expression vector was constructed. The above-mentioned lentiviral expression vector is used together with the viral packaging helper plasmid, the plasmid psPAX2 encoding the viral nucleocapsid proteins Gag / Pol and Rev, and the plasmid pVSVG encoding the viral envelope protein, for the subsequent preparation of different CAR gene-encoded lentiviruses. figure 1 It is the result of identifying viral expression vectors carrying different CAR protein coding sequen...

Embodiment 3

[0195] Example 3. Preparation of CAR gene-encoded virus

[0196] HEK193T was used as packaging cells for the preparation of CAR gene-encoded viruses. HEK293T cells in the logarithmic growth phase were digested, centrifuged at 800 rpm for 5 minutes, and the culture medium was discarded, and then resuspended with DMEM medium (Gbico Company) containing 10% FBS (Gbico Company). After cell counting, adjust the density of the cell suspension to 3.6 × 10 6 / ml, placed in a 37°C cell culture incubator for use. The transfection of the virus packaging plasmid used Lipofectamine 3000 kit (ThermoFisher Company), and operated according to the instructions of the kit. The three plasmids required for lentivirus packaging, including viral vectors containing different CAR genes and the two helper plasmids mentioned in Example 2, were mixed with Lipofectamine3000 according to the ratio recommended in the instructions to form a DNA-liposome complex, and statically placed at room temperature. L...

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Abstract

The invention provides a chimeric antigen receptor for high-efficiency orientation amplification in vitro and an application thereof, and specifically, provides a CAR-encoding molecule having specificselectivity in vitro. Through use of a humanized sorting domain, after infection with target cells, the target cells positive in CAR expression are efficiently sorted through a secondary sorting method so that the in-vitro directional amplification is achieved, the ratio of the CAR-positive target cells in the final product is significantly increased and the process preparation efficiency of CARgene-modified immune cell products is improved. The chimeric antigen receptor provides the table technical assurance for clinical promotion and application of the type of technical products.

Description

technical field [0001] The invention belongs to the field of genetic engineering and immune cell therapy, and relates to a chimeric antigen receptor for high-efficiency directional amplification in vitro and its application. Background technique [0002] Chimeric antigen receptor (CAR) is an artificial receptor molecule composed of antigen-specific antibodies, transmembrane domains, and intracellular signaling co-stimulatory domains. The main feature of this type of molecule is that it can directly recognize target cells through its expression and recognition of related antigen antibody domains, and activate immune cells modified by this type of receptors, such as T cells, through the activation of intracellular signal co-stimulatory domains, Kill target cells directly through cell killing pathways. Compared with the natural T cell killing pathway in the body, CAR-mediated cell killing breaks through the restriction of MHC molecules. The recognition and killing efficiency ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C07K16/2803C07K2317/33C12N2510/00A61K39/464412A61K2239/38A61K2239/31A61K2239/48A61K39/4613A61K39/4631A61K39/4611A61K39/464413C07K2317/622C07K2319/03C07K2319/33A61P35/02C07K14/70517C07K2319/02A61P35/04A61K2039/5158A61K35/17A61K39/0011A61K2039/5156A61K47/65C07K16/2896
Inventor 陈志国赵宇
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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