Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method for membrane surface engineered NK cell, pharmaceutical composition and application

A NK cell and engineering technology, applied in drug combinations, animal cells, vertebrate cells, etc., can solve the problems of non-response to treatment, low transfection efficiency of NK cell viral vector, etc., to enhance the therapeutic effect and enhance tumor infiltration ability. , the effect of eliminating potentially dangerous

Pending Publication Date: 2019-09-06
THE FIRST AFFILIATED HOSPITAL OF BENGBU MEDICAL COLLEGE
View PDF5 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem that the current clinical advanced tumor patients do not respond to the treatment of blocking the PD-1 / PD-L1 pathway, the present invention establishes a preparation system of membrane surface engineered NK cells targeting PD-L1, which enhances the targeting of NK cells on the one hand. Tumor migration ability and killing activity, on the other hand, block the immune escape mediated by the PD-1 / PD-L1 pathway; Aiming at the problem of low transfection efficiency of NK cell virus vectors from peripheral blood, the present invention uses non-viral vectors, Using metabolic sugar bioengineering and copper-free click chemistry to modify PD-L1-targeting polypeptides to the surface of NK cells to enhance the targeting of NK cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method for membrane surface engineered NK cell, pharmaceutical composition and application
  • Preparation method for membrane surface engineered NK cell, pharmaceutical composition and application
  • Preparation method for membrane surface engineered NK cell, pharmaceutical composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of NK-Ac4ManN-P13 cells includes the following steps

[0025] 1.1 NK cells are obtained by in vitro expansion and culture of peripheral blood mononuclear cells, such as figure 1 As shown in, the NK cells (CD3 - CD56 + ) purity up to 95%;

[0026] 1.2 Use L500 medium to adjust the concentration of NK cells to 1×10 6 -3×10 6 a / mL;

[0027] 1.3 Add the targeting carrier Ac4ManNAz or Ac4ManN-DBCO at a final concentration of 26-60 μM, and set the temperature at 37°C with 5% CO 2 Incubate in an incubator for 12-48 hours;

[0028] 1.4 After the incubation, centrifuge at room temperature for 5 minutes at a centrifugal force of 400×g, discard the supernatant, resuspend the cells in L500 medium, and add PD-L1-targeting polypeptide N3-P13 or DBCO-P13 at a final concentration of 10-40 μM Peptides, under the set conditions at a temperature of 37°C containing 5% CO 2 Secondary incubation in the incubator for 0.5-2 hours;

[0029] 1.5 After the second in...

Embodiment 2

[0030] Example 2 Positive detection of NK-Ac4ManN-P13 cells

[0031] 2.1 Prepare NK-Ac4ManN-P13 cells according to the preparation method in Example 1, replace the N3-P13 or DBCO-P13 polypeptide targeting PD-L1 in step 1.4 with N3-P13-FITC or DBCO-P13-FITC polypeptide, and prepare NK-Ac4ManN-P13-FITC cells;

[0032] 2.2 The results of detecting the proportion of NK-Ac4ManN-P13-FITC cells by confocal fluorescence microscopy are as follows: figure 2 As shown in -A, the results of detecting the ratio of NK-Ac4ManN-P13-FITC cells by flow cytometry are as follows figure 2 As shown in -B, the proportion of NK-Ac4ManN-P13-FITC cells reached 100%.

Embodiment 3

[0033] Example 3 In vitro killing activity detection of NK-Ac4ManN-P13 cells

[0034] Detect the killing activity of NK-Ac4ManN-P13 cells prepared in Example 1

[0035]The killing activity of NK-Ac4ManN-P13 cells and control cells with different effect-to-target ratios on FaDu cells of head and neck squamous cell carcinoma was detected by using the real-time label-free cell function analyzer of Essen Bio.

[0036] The specific operation steps are as follows:

[0037] 3.1 Take the FaDu cells in the logarithmic growth phase, and routinely digest them to a concentration of 2.67×10 5 cell suspension per mL;

[0038] 3.2 Add the cell suspension obtained in step 3.1 to the real-time label-free cell function analyzer E-Plate 8 detection plate, add 300 μL to each well, and place in 37°C containing 5% CO 2 Incubate in the incubator for 18-24 hours;

[0039] 3.3 Add 100 μL of NK-Ac4ManN-P13 cell suspension and control group NK cell suspension to E-Plate 8 with different effect-to-...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
purityaaaaaaaaaa
Login to View More

Abstract

The invention relates to the construction of targeted NK cells used for tumor immunotherapy, and specifically relates to a preparation method for a membrane surface engineered NK cell, a pharmaceutical composition and an application. A targeting vector is adopted to introduce an azide group or DBCO on the surface of a NK cell, and polypeptide targeting PD-L1 can be coupled to the surface of the NKcell through copper-free click chemical reaction, so that a membrane surface engineered NK cell can be constructed. An application of the membrane surface engineered NK cell in treating PD-L1 positive tumor including head and neck squamous cell carcinoma, non-small cell lung cancer and triple negative breast cancer is also disclosed. Compared with the prior art, the preparation method has the following advantages: the targeting of the NK cell can be strengthened, so that potential hazards caused by viral vector transfection can be eliminated; the problems of CAR-T cell individualization and complex manufacturing processes can be solved; and the tumor infiltration capabilities and targeted killing activity of the NK cell can be enhanced, and immune escape mediated by a PD-1 / PD-L1 pathway can be effectively blocked, so that the membrane surface engineered NK cell has potential clinical application prospets.

Description

technical field [0001] The invention relates to a targeted NK cell construction for tumor immunotherapy, in particular to a preparation method, pharmaceutical composition and application of membrane surface engineered NK cells. Background technique [0002] PD-1 / PD-L1 immune checkpoint inhibitors can activate significant and long-lasting anti-tumor immune responses, and have been approved for many cancers, including advanced non-small cell lung cancer, squamous cell carcinoma of the head and neck, and triple-negative breast cancer. It is currently the most promising tumor treatment method. Unfortunately, about 70% of patients with advanced tumors do not respond to PD-1 / PD-L1 immune checkpoint inhibitor therapy. Therefore, it is important to explore how to improve the therapeutic effect of blocking the PD-1 / PD-L1 pathway Meaning is also a hot topic of current research. [0003] As an important part of innate immunity, NK cells have a direct killing effect on tumor cells, an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0646A61K35/17A61P35/00
Inventor 赵报马士崟韩跃峰李坤张明洁李慧周兰柱崔忆璇
Owner THE FIRST AFFILIATED HOSPITAL OF BENGBU MEDICAL COLLEGE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com