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Chimeric antigen receptor capable of specifically activating NK cells and application thereof

A chimeric antigen receptor and NK cell technology, applied in the field of biomedicine, can solve problems such as lack of research

Active Publication Date: 2019-12-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The structure of chimeric antigen receptors in CAR-NK currently used in clinical practice mainly follows the structure of the second and third generation of CAR-T. At present, there is still a lack of research on the structural modification of CAR for NK cell activation signal transduction.

Method used

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  • Chimeric antigen receptor capable of specifically activating NK cells and application thereof
  • Chimeric antigen receptor capable of specifically activating NK cells and application thereof
  • Chimeric antigen receptor capable of specifically activating NK cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction and stability testing of CAR-NK cell lines

[0056] experimental method:

[0057] 1. CAR lentiviral packaging: clone the chimeric antigen receptor sequence into a lentiviral vector, transfect the Lenti-X-293 cell line together with the packaging plasmid, culture continuously for 72 hours, collect the virus supernatant and filter it. The virus was concentrated by centrifugation at 20000rpm (82700g) for 2.5 hours, and the titer was quantified after the virus particles were resuspended.

[0058] 2. Establishment of CAR-NK cell line: Infect NK-92 cells with lentivirus according to MOI=1-50, and use FLAG-APC flow cytometry antibody to sort CAR-positive cells; secondary sorting. The secondary sorting cells are divided into single clones, and expanded culture.

[0059] 3. Stability detection of CAR-NK cells: CAR-NK cells were continuously cultured and passaged in complete medium, and the positive rate of CAR in the established cells was detected by F...

Embodiment 2

[0062] Example 2: CAR Opti The structure significantly improves the killing activity of NK cells

[0063] experimental method:

[0064] Cell killing experiment: The effect-to-target ratio (E:T) of CAR-NK cells is generally 1:1 and 1:2. The killing time is 2hrs. The target cells are HCC cell lines, which are all adherent cells and need to be prepared into a single cell suspension. Before cell digestion, PBS was used to wash off excess medium, and 0.25% trypsin was added to digest for 2-5min. Count effector cells: According to the effector-target ratio, the number of effector cells per tube or well is 40,000 and 80,000; take a certain amount of CAR-NK cells, centrifuge to resuspend and count, and adjust the concentration to 40,000cell / 250μL or 80,000cell / 250μL. Target cells were labeled with CSFE (sigma, 150347), every 10 6 Add 10 μL of 50 μM CSFE to each target cell, and incubate at 37 degrees for 15 minutes in the dark. Wash twice with ice-cold PBS, resuspend the cells w...

Embodiment 3

[0067] Example 3: CAR OptiStudy on the activation of signaling pathways related to NK cell killing by structure

[0068] experimental method:

[0069] Western-blot: Take 5×10 NK cells after killing 6 , 3000rpm, centrifuge for 5 minutes, transfer to a 1.5mL EP tube; add 200μL protein extraction reagent (Thermo ScientificPierce, 78505) to the cell pellet to resuspend, ice-bath for 30 minutes, shake once every 5 minutes; After 1 minute, the supernatant was carefully transferred to a clean EP tube, and protein quantification was performed using the BCA protein quantification kit (Thermo Scientific Pierce, A53225). After quantification, adjust the protein concentration of each group of samples to be consistent, add 5× loading buffer (Biyuntian, P0015) to the protein sample, mix well, boil in a water bath at 100°C for 5 minutes, cool and load the sample (10-20μL protein / lane ), while loading the protein Marker (Invitrogen, 26625); use 12% SDS polyacrylamide gel electrophoresis to...

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Abstract

The invention relates to the field of biological medicines, in particular to a chimeric antigen receptor specifically optimized according to NK cell signal transduction characteristics and an application thereof. The chimeric antigen receptor scFv is composed of a light chain variable region and a heavy chain variable region and connected to an NKp44 transmembrane region through a CD8 hinge structure, and an intracellular segment activation signal transduction structural domain is composed of a CD3 zeta ITAM motif, a 2B4 activation motif, a DNAM1 activation motif and a DAP10 activation motif which are connected in series. The chimeric antigen receptor is used for modifying natural killer (NK) cells, and the modified NK cells (CAR-NK) can be used for treating tumors with positive tumor specific antigens. In a killing test, compared with a classic third-generation T-CAR structure, the structure has the advantages that the killing capability of the NK cells on tumor cells is obviously enhanced, and good anti-tumor activity is shown in an in-vivo model.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric antigen receptor improved according to the NK cell activation signal transduction mechanism and its application. Background technique [0002] Since the invention of CAR-T cell therapy (CTL-019) by Professor Carl June of the University of Pennsylvania, many biotechnology companies in the world have carried out further exploration of CAR-T. The first three giants to develop CAR-T cell therapy were Novartis, KitePharma and JunoTherapeutics. The initial research on CAR-T focused on blood-related tumors. With the deepening of research, more new targets, new technologies, and new therapeutic areas are being explored one after another. While achieving exciting clinical trial efficacy, safety has also become a major problem that plagues the progress of CAR-T: cytokines Storms, off-target effects, severe allergic reactions, and neurotoxicity have seriously limited the widespread ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61K2039/5158A61P35/00C07K14/7051C07K16/303C07K2317/622C07K2317/64C07K2319/02C07K2319/03C07K2319/33C12N5/0646C12N15/86C12N2510/00C12N2740/15043
Inventor 王全兴郭猛刘艳芳刘芳丁国善曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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