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Targeted folate receptor alpha chimeric antigen receptor and application thereof to preparation of medicine for preventing or treating malignant tumors

A technology of chimeric antigen receptors and folate receptors, which is applied in the field of chimeric antigen receptors targeting folic acid receptor α in the preparation of drugs for the prevention or treatment of malignant tumors. question

Active Publication Date: 2019-05-24
重庆福美干细胞生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, primary NK cells have the same defects as primary T cells. For example, the expansion ability of NK cells in different patients varies significantly.
In addition, the exogenous gene transfection efficiency of primary NK cells is low and the survival time is limited, and the effect of CAR-loaded cells needs to be improved

Method used

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  • Targeted folate receptor alpha chimeric antigen receptor and application thereof to preparation of medicine for preventing or treating malignant tumors
  • Targeted folate receptor alpha chimeric antigen receptor and application thereof to preparation of medicine for preventing or treating malignant tumors
  • Targeted folate receptor alpha chimeric antigen receptor and application thereof to preparation of medicine for preventing or treating malignant tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of a Chimeric Antigen Receptor Expression Vector Targeting Folate Receptor α

[0048] A chimeric antigen receptor targeting folate receptor α (abbreviated as αFR-CAR) expression vector was constructed.

[0049] like figure 1 As shown, αFR-CAR is composed of human CD8α signal peptide, polypeptide binding to folate receptor α, human immunoglobulin G1Fc segment, human CD8α hinge region, human CD28 transmembrane segment, human CD28 intracellular segment, human CD137 cell Inner segment and human CD3ζ intracellular segment.

[0050] Wherein, the coding nucleotide sequence of the human CD8α signal peptide is shown in SEQ ID NO.1, specifically:

[0051] ATGGCCCTCCCCAGTTACCGCCCTTCTCCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCC.

[0052] Folate receptor alpha (αFR) binding polypeptides are single chain variable regions (scFv) of antibodies raised against folate receptor alpha (αFR). The single-chain variable region (scFv) of the anti-folate receptor alpha (αFR) ...

Embodiment 2

[0099] Example 2 Preparation of Lentivirus for Expression of Chimeric Antigen Receptor Targeting Folate Receptor α

[0100] 1. The equipment and materials are shown in Table 1 below.

[0101] Table 1 Equipment and materials required for expressing lentiviral vectors

[0102] Reagents and materials

company

Item No.

DMEM medium

Gibco

11960044

Fetal bovine serum (FBS)

Gibco

16140

1X D-PBS

Beyotime

C0221D

PolyJet TM DNATransfection Reagent

SignaGen

SL100688

Disposable syringe filter 0.45um

Millipore

SLHV033RB

Ultra-clear SW28 centrifuge tube

Beckman

L90K high speed centrifuge with SW28 rotor

Beckman

293T cell line

FuHeng BioLogy

FH0823

[0103] 2. Specific operation steps:

[0104] 2.1. Packaging of lentivirus:

[0105] After the cultured 293T cells were digested with trypsin and washed, the cells were resuspended with DMEM complete mediu...

Embodiment 3

[0121] Example 3 Construction of Chimeric Antigen Receptor Modified NK-92 Cell Targeting Folate Receptor α

[0122] The chimeric antigen receptor targeting folate receptor α prepared in Example 2 was used to infect NK-92 cells with lentivirus, and the specific operations were as follows:

[0123] 1. Inoculate NK-92 cells in a 24-well plate, 5×10 per well 6 cells.

[0124] 2. Add 200U / ml interleukin-2 to each well to maintain cell proliferation.

[0125] 3.5% CO 2 , and cultured at 37°C for 24 hours.

[0126] 4. Add 50 μl of the folate receptor α-targeting chimeric antigen receptor-expressing lentivirus concentrate prepared in Example 2 and 5 mg / ml polybrene to the NK-92 cell culture wells, and mix well.

[0127] 5.5% CO 2 , and cultured overnight at 37°C.

[0128] 6. Remove the old medium supernatant and add fresh NK-92 cell complete medium containing 200 U / ml interleukin-2.

[0129] 7.5% CO 2 , and cultured at 37°C for 3 days.

[0130] 8. Add 5 μg / ml puromycin to the...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a targeted folate receptor alpha chimeric antigen receptor and an application thereof to preparation of a medicinefor preventing or treating malignant tumors. The targeted folate receptor alpha chimeric antigen receptor sequentially comprises a human CD8 alpha signal peptide, a polypeptide binding a folate receptor alpha, a human immune globulin G1Fc segment, a human CD8 alpha hinge area, a human CD28 transmembrane segment, a human CD28 Notch intracellular domain, a human CD137 Notch intracellular domain anda human CD3 zeta Notch intracellular domain. The chimeric antigen receptor is led into an NK-92 cell, and a folate receptor alpha positive human tumor cell line and a human ovarian cancer transplantedtumor model can be specifically recognized and killed. The chimeric antigen receptor is safe, specific and effective, the NK-92 cell is modified, and the chimeric antigen receptor has great application potential in clinical treatment of folate receptor alpha positive malignant tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chimeric antigen receptor targeting folic acid receptor α and its use in the preparation of drugs for preventing or treating malignant tumors. Background technique [0002] With the development of tumor immunology theory and technology, the role of adoptive cell therapy (ACT) in immunotherapy in tumor treatment has been paid more and more attention. In particular, the chimeric antigen receptor (CAR) technology that has emerged in recent years has made rapid progress. CAR-modified immune cells have shown significantly better tumor killing activity than traditional ACT in vitro and clinical trials, which has triggered a new era of tumor therapy. A new wave of translational research. [0003] CAR usually contains a single-chain variable fragment (scFv) targeting and recognizing tumor antigens (derived from antibodies) and different intracellular signaling domains (derived...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K35/17A61P35/00
CPCA61K39/4631A61K39/4613A61K39/464402
Inventor 李伟强邵文陶冉启平周兴
Owner 重庆福美干细胞生物科技发展有限公司
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