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Bispecific NK cell agonist as well as preparation method and application thereof

A technology of NK cells and cells, applied in the field of biomedicine, can solve the problem of limited NK cells ability

Active Publication Date: 2022-04-15
SHANGHAI NK CELLTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are still few research reports on NK cell agonists, and the existing NK cell agonists have limited ability to activate NK cells, which still needs to be further improved

Method used

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  • Bispecific NK cell agonist as well as preparation method and application thereof
  • Bispecific NK cell agonist as well as preparation method and application thereof
  • Bispecific NK cell agonist as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Construction of fusion protein expression vector of bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL

[0096] 1. Obtain scfv-CD16A-Trimer-4-1BBL fusion gene

[0097] Obtain the sequence information of scfv-CD16A (scfv-CD16A includes V H and V L , and V H and V L connected through linker1, where V H The amino acid sequence is shown in SEQ ID NO: 1, V L The nucleotide sequence of linker1 is shown in SEQ ID NO:2, the nucleotide sequence of linker1 is shown in SEQ ID NO:3), obtain 4-1BBL extracellular segment 71-254 sequence from NCBI (as shown in SEQ ID NO: 5), scfv-CD16A is connected to three 4-1BBL extracellular segments through linker2 (as shown in SEQ ID NO: 4), and the three 4-1BBL extracellular segment domains are sequentially passed through linker3 (as shown in shown in SEQ ID NO:6) and linker4 (shown in SEQ ID NO:7) are connected. The original scfv-CD16A-Trimer-4-1BBL fusion gene obtained without codon optimization is shown in SEQ ID NO:9.

[...

Embodiment 2

[0103] Example 2. Expression and purification of the fusion protein of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL

[0104] 1. Fusion protein expression

[0105] The positive clone constructed in Example 1 was expanded and cultured in 50 mL LZ liquid medium, and the plasmid PGAP-zα-scfv-CD16A-Trimer-4-1BBL was extracted in a large amount using a kit (Axygen Company). The expression plasmid PGAPzα-scfv-CD16A-Trimer-4-1BBL prepared in the previous step was linearized with endonuclease SpeI and recovered by ethanol precipitation.

[0106] Ethanol precipitation steps: 1) Add twice the volume of absolute ethanol and 0.1 times the volume of 3M sodium acetate to the enzyme digestion reaction system, and precipitate at -20°C for more than 2 hours. 2) Centrifuge at 12000 g for 10 minutes, discard the supernatant. 3) The pellet was resuspended in 300 μL of 70% ethanol, centrifuged at 12000 g for 10 minutes, and the supernatant was discarded. 4) Dry at 37°C, resuspend in d...

Embodiment 3

[0120] Example 3. Functional verification of the fusion protein of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL inducing NK cell activation in vitro

[0121] Human PBMCs were isolated by Ficoll density gradient centrifugation. Dilute the isolated PBMC to 1.0×10 with RPMI1640 containing 10% FBS 6 individual / mL. Add 10 mL of diluted cells to the T25 culture flask. After 12 hours of incubation, the scFv-CD16A-Trimer-4-1BBL fusion protein was added to the flasks at a final concentration of 20 nM. After 24 hours of culture, the phenotype changes of NK cells were detected by flow cytometry.

[0122] The result is as Figure 5 As shown, compared with the PBS control group, in the experimental group added scfv-CD16A-Trimer-4-1BBL fusion protein, the main activating molecules on the surface of NK cells such as CD69, NKp30 and killing molecules 4-1BB, TRAIL, GranzymeB, The expression of chemotactic molecule CX3CR1 was significantly up-regulated, so it was judged that NK ...

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Abstract

The invention relates to the field of biological medicine, in particular to a bispecific NK cell agonist as well as a preparation method and application thereof. The invention provides a fusion protein, which takes a CD16A receptor and a 4-1BB receptor as targets to obtain a novel bispecific NK cell agonist drug, namely scfv-CD16A-Trimer-4-1BBL, which has the activity of activating and amplifying NK cells in vitro, enhances the antiviral and anti-tumor capabilities of the NK cells, has potential effects of promoting NK cell proliferation and enhancing T cell functions, and can be used for preparing a novel bispecific NK cell agonist drug. The application potential of in-vitro culture and amplification of the NK cells is achieved, and the amplified NK cells have good cytotoxicity.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a bispecific NK cell agonist and its preparation method and application. Background technique [0002] NK cells are the main effector cells of the innate immune system. Compared with T cells, NK cells can kill tumor cells and virus-infected cells without MHC restriction by using perforin, granzyme and related mechanisms without prior stimulation. NK cells express many activating receptors, which can effectively recognize the stress ligands produced by tumor cells or infected cells, thereby effectively killing tumor cells, but at the same time, tumor cells also secrete and produce many immunosuppressive molecules, forming immunosuppression The tumor microenvironment can inhibit the activity of NK cells and the anti-tumor ability in this environment; make NK cells in a state of exhaustion. At present, there are also many strategies to restore the exhaustion of NK cells in the tumor micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19A61K38/17A61K39/395A61P31/04A61P35/00A61P35/02A61P31/14A61P31/16A61P31/20C12N5/0783C12R1/84
CPCA61K38/17A61P31/04A61P31/14A61P31/16A61P35/00A61P35/02A61P31/20C07K19/00C12N5/06C12N15/62C12R2001/84C07K14/70578C07K14/70575C12N5/0646C12N15/815C07K16/283C07K2319/00C07K2317/622C07K2317/74A61P29/00A61P31/12C12N2800/22C12N2501/998A61K38/00C07K2317/732C07K2319/33C07K2319/75
Inventor 肖卫华田志刚李洋阳谢思奇陈敏华
Owner SHANGHAI NK CELLTECH CO LTD
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