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Expansion method of various lymphocyte subpopulations and application of expansion method

A technology of lymphocytes and subgroups, applied in the field of biomedical engineering, can solve the problems of large differences in the maintenance of T cells, expensive and time-consuming methods for expanding immune cells, etc.

Active Publication Date: 2016-06-01
杭州朔溪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] For this reason, the technical problem to be solved by the present invention lies in the fact that existing methods for expanding immune cells are expensive and time-consuming, and peripheral blood mononuclear cells come from multiple donors, and the maintenance effect on T cells is quite different, so A method for stimulating peripheral blood in vitro to efficiently and simultaneously expand multiple lymphocyte subsets and the prepared cell expansion kit

Method used

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  • Expansion method of various lymphocyte subpopulations and application of expansion method
  • Expansion method of various lymphocyte subpopulations and application of expansion method
  • Expansion method of various lymphocyte subpopulations and application of expansion method

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Embodiment 1

[0053] Immune cell subset analysis: In order to study the influence of the expansion methods of various lymphocyte subsets on the expansion of immune cell subsets, a method for simultaneous analysis of various immune cell subsets by multi-color flow cytometry was first established . Expanded immune cells were stained with 6 different antibodies. For staining, cells were resuspended in 100 μl of MACS buffer and stained with antibody mixture for 15 min at 4°C in the dark. Cells were stained with MACS buffer before and after staining. The antibodies used included: FITC-labeled anti-Vdelta2 T cell receptor antibody, APC-labeled anti-CD3 antibody, PE-labeled anti-CD4 antibody, PE-CyC7-labeled anti-CD56 antibody, APC-CyC7-labeled anti-CD16 Antibody, V450-labeled anti-CD8a antibody. At the same time, AcuriC6 flow cytometer was used for single-color or double-color analysis, and the antibodies used included FITC-anti-Vdelta2 T cell receptor antibody, APC-anti-CD3 antibody and PE-anti...

Embodiment 2

[0055] Preliminary expansion of immune cells: adding IFNγ at a concentration of 500-1500 IU / ml to the PBMC medium collected from the blood of tumor patients, preferably 1000 IU / ml in this embodiment, and zoledronic acid at a concentration of 2-10 μM, In this embodiment, it is preferably 5 μM. After culturing for one day, add an OKT3 antibody with a concentration of 20-100 ng / ml. The tumor includes hematological tumors and solid tumor cells. In this embodiment, it is preferably 50 ng / ml and the concentration is 100- 600IU / mL of recombinant human interleukin-2, preferably 300IU / mL in this embodiment, stimulates peripheral blood mononuclear cells for 7-14 days, and the initial expansion is a conventional CIK immune cell expansion method combined with zoledrone The combined use method of acid (ZOL), referred to as CIK+ZOL for short, said CIK+ZOL compared with the independent CIK method and ZOL method, increased the number of expansion of the total number of immune cells, such as ...

Embodiment 3

[0057] On the basis of Example 2, adding IL-15 with a concentration of 10ng / ml and IL-18 with a concentration of 10ng / ml to further increase the ratio of CD3CD56 double-positive cells, with or without IL-15 and IL-18 The changes of γδT, CD3+CD56+T, CD3+CD8+T, NK, CD16 high expression and CD56 high expression, CD16 low expression and CD56 high expression cells in the PBMC samples collected from the blood of tumor patients were initially expanded for 14 days. Figure 15-20As indicated, data are presented as mean ± SD, *, **: P<0.05 or 0.01. IL-15 is a cytokine similar in structure to IL-2, which can regulate the activation and proliferation of T cells and natural killer NK cells, and interleukin-18 (IL-18) is a cytokine belonging to the IL-1 family , IL-18 can enhance the maturation of T cells and NK cells, cytokine production, and cytotoxicity, together with IL-15, IL-18 can stimulate NK cells and T cells to release interferon gamma (IFN-γ).

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Abstract

The invention discloses an expansion method of various lymphocyte subpopulations and application of the expansion method to preparation of an adoptive immunotherapy medicine for cancers. The expansion method comprises the following steps: S1, adding IFN gamma and zoledronic acid into a culture medium of PBMC collected in the blood of a tumor patient, and re-adding an OKT3 antibody and recombinant human interleukin-2 after culture to stimulate PBMC so as to finish preliminary expansion; S2, after finish of preliminary expansion, mixing immune lymphocytes with feeder layer cells, and after adding zoledronic acid, an OKT3 antibody and recombinant human interleukin-2, continually performing expansion. By adoption of the expansion method, after twice expansion, the sum of the immune cells can be 10,000 to 80,000 times by expansion, the expanded immune cells include alpha beta T cells, gamma delta T cells, NKT cells and NK cells, and tumor cells can be directly killed, or a cellular immunotherapy drug is prepared to kill the tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a method for efficiently and simultaneously expanding multiple lymphocyte subsets and a prepared cell expansion kit. Background technique [0002] Adoptive immunotherapy is a treatment approach that has recently shown promising results in clinical trials for solid tumors and hematologic cancers and is a rapidly growing field of medical research. Adoptive immunotherapy methods include isolation of immune cells, ex vivo cell expansion, and reinfusion of the expanded immune cells into cancer patients. Examples of successful eradication of cancer by adoptive immunotherapy include the application of tumor-infiltrating lymphocytes (TIL), T cell receptor (TCR)-modified T cells, and chimeric antigen receptor (CAR)-modified T cells. These immune cells are mainly αβT cells. Expression of alpha and beta chains make up the T cell receptor. In addition to αβT cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61K48/00A61P35/00
Inventor 卓朗王晖王柯
Owner 杭州朔溪生物医药有限公司
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