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Enhanced type anti-tumor NK cell and preparation method and application thereof

An NK cell and anti-tumor technology, applied in the biological field, can solve the problems of loss and impaired NK cell cytotoxicity, and achieve the effects of small immune response, strong tumor cell lethality, and large gene fragments

Active Publication Date: 2018-05-01
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, shedding proteins of NKG2DL (NKG2DLs for short) lead to impaired NK cell cytotoxicity due to loss of antigenic NKG2DL and NKG2DLs-induced downregulation of NKG2D

Method used

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  • Enhanced type anti-tumor NK cell and preparation method and application thereof
  • Enhanced type anti-tumor NK cell and preparation method and application thereof
  • Enhanced type anti-tumor NK cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Synthesis of genes encoding dual targeting chimeric receptors

[0035] Through the method of whole gene synthesis, a 450bp PD-1 extracellular segment (21-170aa), a 648bp NKG2D full-length segment (1-216aa), and a 126bp co-stimulatory molecule 41BB intracellular segment (214-255aa) were synthesized. ), and connect them in series to obtain the PD1-NKG2D-41BB gene fragment.

[0036] 1. Primer design

[0037] PD1-NKG2D-41BB is a DNA molecule containing 1266bp. 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 13 pairs of upstream ...

Embodiment 2

[0068] Example 2: Construction of recombinant lentiviral vector

[0069] 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- The CopGFP lentiviral vector and the digested PD1-NKG2D-41BB gene fragment were incubated overnight at 16°C using the 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.

[0070] 1. Double digestion pCDH-CMV-MCS-EF1-CopGFP lentiviral vector

[0071] The enzyme digestion reaction system is as follows (100μl):

[0072]

[0073] Reaction conditions for enzyme cleavage: react at 37°C for 3 hours.

[0074] 2. Double digestion of PD1-...

Embodiment 3

[0081] Example 3: Packaging of lentivirus

[0082] 1. Extraction of lentiviral packaging plasmid and target plasmid

[0083] 1.1 Plasmid transformation

[0084] 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 add 800 μL of LB medium respectively, mix thoroughly, place in a 37° C. constant temperature shaking incubator, and shake at 100 rpm / min for 1 hour.

[0085]After the cultivation, 200 μL of the bacterial liquid was taken and spread on three LB solid medium (containing ampic...

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Abstract

The invention discloses an enhanced type anti-tumor NK cell and a preparation method and application thereof. The anti-tumor NK cell is an NK cell modified by a dual-targeting chimeric receptor, and the dual-targeting chimeric receptor is composed of a PD-1 extracellular domain, an NKG2D full-length domain and a costimulatory molecule 41BB intracellular domain. The anti-tumor NK cell disclosed bythe invention has the advantages that by adopting the dual-targeting chimeric receptor for modifying the NK cell, the anti-tumor treatment effect of the NK cell can be improved, specific recognition and killing of solid tumors can be realized, and excessive immune response of an organism can not be caused.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to an enhanced 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-...

Claims

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Application Information

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