Culture method for simultaneously amplifying gamma delta T and NK

A medium and co-cultivation technology, applied in the field of immunomedicine, can solve the problem of inability to use allogeneic reinfusion, achieve excellent anti-tumor effect, save time and cost of preparation, and reduce GVHD.

Pending Publication Date: 2021-09-24
GUANGZHOU BIO GENE TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Culture method for simultaneously amplifying gamma delta T and NK
  • Culture method for simultaneously amplifying gamma delta T and NK
  • Culture method for simultaneously amplifying gamma delta T and NK

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Embodiment 1

[0054] Example 1: Preparation of Artificial Antigen Presenting Cells (aAPC)

[0055] 1. Construction of PB-mIL15-CD137L-Puro vector: Membrane-type IL15 (mIL15) is connected to CD137L through a self-cleaving 2ALinker. The 2A Linker expresses 20 amino acids and can self-cleavage at the 19+1 site, wherein mIL15 The first 19 amino acids remain. Its structure is CMV-mIL15-2A-CD137L-EF1-GFP-Puro, the vector contains GFP green fluorescent protein, which is used to indicate the integration of mIL15 and CD137L genes in the cell genome; the included puromycin (Puro) resistance as drug screening markers. The mIL15-CD137L sequence was inserted between BamHI and NotI. The obtained carrier map is as figure 1 mentioned.

[0056] 2. Among them, mIL15 (membrane IL15) is anchored on the surface of K562 cell membrane through the hinge region and transmembrane region, and exerts its function by combining with IL15R on T cells. Its structure is as figure 2 shown.

[0057] 3. The CD137L mol...

Embodiment 2

[0063] Example 2: Culture of γδT and NK Mixed Cells (NK / γδT)

[0064] 1. Separation of PBMCs: Collect 50ml of peripheral blood, and add 15ml of lymphocyte separation solution to two 50ml sterilized centrifuge tubes under the normal working condition of the ultra-clean workbench. Slowly inject the peripheral whole blood into the supernatant of the lymphocyte separation liquid in 2 centrifuge tubes, and add 25-30ml of peripheral blood to each centrifuge tube. Centrifuge at 700g×20 minutes, increase speed 1, decrease speed 2, room temperature. After centrifugation, blood is separated into 4 layers consisting of plasma (upper layer), mononuclear cells between plasma and separation fluid (layer 2), separation fluid (layer 3), and erythrocyte layer (bottom layer). Collect the layer 2 mononuclear cells into a new centrifuge tube with a pipette. Add 20ml of PBS to dilute the cell suspension, and centrifuge at 500g for 10min.

[0065] 2. Stimulate γδT and NK cells on D0: remove the ...

Embodiment 3

[0069] Embodiment 3: γδT and NK ratio detection

[0070] Samples were taken on Day0, Day7, Day10, and Day14 of the culture, stained with anti-human CD3 antibody, anti-human CD56 antibody, anti-human TCRαβ antibody, anti-human TCRγδ antibody, anti-human TCRVδ1 antibody, and anti-human TCRVδ2 antibody, and flow cytometric detection of NK cells and γδT cell expression.

[0071] The results are shown in Table 2 and image 3 As shown, when the cells in group 1 were expanded to 14 days (D14), the main cell subpopulation was γδT cells, accounting for 94.2%; groups 2, 3, and 4 could effectively expand γδT and NK cell subpopulations simultaneously, About 40-65%; Group 2 and Group 3 can obtain relatively balanced γδT and NK subgroups, and Group 4 has more NK ratio. γδT and NK ratios can stimulate different ratios of cell subtypes by adjusting the number of aAPCs. In addition, the four culture schemes only contain a very low proportion of αβT cells (less than 1%), and are all suitable...

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Abstract

The invention relates to the field of immune medicine, in particular to a cell culture method which comprises the following steps: co-culturing an inactivated artificial antigen presenting cell with high expression of mIL15 and CD137L and PBMC in a culture medium, wherein the culture medium comprises a basic culture medium and additional components, the additional components comprise IL2 with the concentration of 100 IU/mL to 1200 IU/mL and zoledronic acid with the concentration of 3 mu M to 7 mu M. The ratio of NK cells to gamma delta T cells cultured by the method is high, and the cells have a more excellent anti-tumor effect.

Description

technical field [0001] The invention relates to the field of immunomedicine, in particular to a culture method for simultaneously amplifying γδT and NK. Background technique [0002] At present, CAR T cells have made significant progress in the treatment of tumors, especially CD19 CAR T cells have achieved encouraging results in the treatment of B-cell malignant hematomas. Most of the current CAR T clinical trials are modified using the patient's own T cells. Therefore, the decline in the quality and quantity of T cells and the increase in the time and cost of preparing autologous CAR T cell products have become important factors hindering the development of CAR T cell therapy. . Therefore, the invention of universal CAR T cell products can not only improve the quality and quantity of CAR T cells, but also save production time and cost. In addition, traditional CAR T is modified by using αβT cells, which rely on HLA to recognize tumor-associated antigens. If αβT cells are ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N5/0646C12N2501/2315C12N2501/2302C12N2501/2321C12N2501/999C12N2502/99
Inventor 李光超陈卓莹罗敏丁雯陈雅南
Owner GUANGZHOU BIO GENE TECH CO LTD
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