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Novel anti-tumor NK cell as well as preparation method and application thereof

A NK cell and anti-tumor technology, applied in the biological field, can solve problems that threaten the lives of patients, and achieve the effect of wide treatment range, small immune response, and large capacity for exogenous target gene fragments

Active Publication Date: 2018-05-18
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, despite the continuous progress in the research and development of CAR-T technology, there are still many challenges. One of the main obstacles is that although CAR-T has a strong ability to kill cancer cells, the excessive immune response caused by itself has the potential to Threatening the patient's life
Moreover, immune cell technologies such as CAR-T or TCR-T (T cell receptor chimeric T cells) have so far been seldom effective in the treatment of solid tumors, and the treatment of solid tumors is the real main battlefield for anti-tumor

Method used

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  • Novel anti-tumor NK cell as well as preparation method and application thereof
  • Novel anti-tumor NK cell as well as preparation method and application thereof
  • Novel anti-tumor NK cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Synthesis of chimeric co-stimulatory switch receptor encoding gene

[0036] Through the method of whole gene synthesis, a 450bp PD-1 extracellular segment (21-170aa), a 270bp NKG2D transmembrane and intracellular segment (44-132aa), and a 126bp co-stimulatory molecule 41BB intracellular segment ( 214-255aa), and connect them together to obtain the PD1-NKG2D-41BB gene fragment.

[0037] 1. Primer design

[0038] PD1-NKG2D-41BB is a DNA molecule containing 849bp. Every 60bp is a fragment, and there must be 10bp overlap between every two fragments, for example, 1-60bp is fragment 1, 50-110bp is fragment 2, 100-160bp is fragment 3, and so on. Design an upstream primer at the 5' end of Fragment 1, named as F 1 ; Design a downstream primer at the 3' end of fragment 2, named as R 1 ; Design an upstream primer at the 5' end of fragment 3, named as F 2 ; Design a downstream primer at the 3' end of fragment 4, named as R 2 , and so on. There are 9 pairs of upstr...

Embodiment 2

[0062] Example 2: Construction of recombinant lentiviral vector

[0063] Cloning the PD1-NKG2D-41BB gene fragment obtained in Example 1 into the pCDH-CMV-MCS-EF1-CopGFP lentiviral vector, such as figure 1 shown. The pCDH-CMV-MCS-EF1-CopGFP lentiviral vector and the PD1-NKG2D-41BB gene fragment were respectively digested with restriction endonucleases XbaI and EcoR I to obtain the linearized pCDH-CMV-MCS-EF1- CopGFP lentiviral vector and digested PD1-NKG2D-41BB gene fragment were incubated overnight at 16°C using T4 DNA ligase system. Then transform the competent cells, screen the positive colonies, and extract the plasmids of the positive colonies to obtain the PD1-NKG2D-41BB-pCDH expression vector.

[0064] 1. Double digestion pCDH-CMV-MCS-EF1-CopGFP lentiviral vector

[0065] The enzyme digestion reaction system is as follows (100μl):

[0066]

[0067] Reaction conditions for enzyme cleavage: react at 37°C for 3 hours.

[0068] 2. Double digestion of PD1-NKG2D-41BB g...

Embodiment 3

[0075] Example 3: Packaging of lentivirus

[0076] 1. Extraction of lentiviral packaging plasmid and target plasmid

[0077] 1.1 Plasmid transformation

[0078] Take one 100 μL Stbl3 competent cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and two 100 μL Escherichia coli TOP10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.); Plasmid PD1-NKG2D-41BB-pCDH was added to Stbl3 competent cells, and 1 μg of lentiviral packaging helper plasmids pSPAX2 and PMD2G were respectively added to 100 μL of E. coli TOP10 competent cells. Incubate on ice for 30 minutes, then immediately heat shock in a 42°C water bath for 90 seconds, and place in an ice bath for 2 minutes. Then, 800 μL of LB medium were added, mixed well, placed in a constant temperature shaking incubator at 37°C, and shaken at 100 rpm / min for 1 hour.

[0079] After the cultivation, 200 μL of the bacterial liquid was taken and spread on three LB solid medium (containing ampicillin...

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Abstract

The invention discloses a novel anti-tumor NK cell as well as a preparation method and application thereof. The anti-tumor NK cell is an NK cell transformed by a chimeric co-stimulatory transformationreceptor; the chimeric co-stimulatory transformation receptor consists of a PD-1 extracellular fragment, an NKG2D transmembrane and intracellular fragment and a co-simulatory molecular 41BB intracellular fragment. The novel anti-tumor NK cell disclosed by the invention has the beneficial effects that the chimeric co-stimulatory transformation receptor is adopted for transforming the NK cell, so that the anti-tumor treating effect of the NK cell can be improved, the specific recognition and killing for solid tumor are realized, and no extreme immunized response is caused to an organism.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a novel anti-tumor NK cell and its preparation method and application. Background technique [0002] In recent years, tumor immune cell therapy has attracted much attention in tumor treatment due to its remarkable curative effect, especially T cells based on chimeric antigen receptor (CAR-T), which have shown good clinical treatment in hematological malignancies. The targeting, lethality and persistence of the drug make it the most convincing breakthrough in the field of tumor immunotherapy. However, although the research and development of CAR-T technology has made continuous progress, it still faces many challenges. One of the main obstacles is that although CAR-T has a strong ability to kill cancer cells, the excessive immune response caused by itself has the potential to Threatening the patient's life. Moreover, immune cell technologies such as CAR-T or TCR-T (T...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867C12N15/66A61K35/17A61P35/00
CPCA61K35/17C07K14/70521C07K2319/03C12N5/0646C12N15/66C12N15/86C12N2510/00C12N2740/15043
Inventor 朱武凌路臣桂张会勇李冬贝郭长江李明凤夏文姣王晓银吕探宇
Owner XINXIANG MEDICAL UNIV
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