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
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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

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  • 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-...

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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

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Application Information

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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
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