Method for simultaneously inducing and amplifying V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells

A technology of CD3-CD56 and NK cells, which is applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of micrometastasis, dose-dependent toxicity, radiotherapy insensitivity, etc. The effect of high expression of NKG2D activating receptor, simple and easy-to-operate method, and high purity of effector cells

Active Publication Date: 2015-02-18
HRYZ (SHENZHEN) BIOTECH CO +1
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Each of the three therapies has limitations: surgical treatment cannot remove the micrometastases of the tumor, and it is difficult to obtain a radical curative effect; radiotherapy has dose-dependent toxicity and cannot effectively remove potential metastases. In addition, some patients are resistant to radiotherapy due to radiation resistance. Insensitive, chemotherapy can easily cause systemic immunosuppression, and in some patients, chemotherapy drugs cannot effectively enter the tumor tissue or patients develop resistance to chemotherapy drugs, which affects the curative effect
[0005] In immune cell therapy, a single type of cells is mainly cultivated. For the simultaneous cultivation of multiple types of immune cells, since the culture conditions applicable to each type of cells are different from each other, it is necessary to simultaneously parallelize multiple cell types. Carry out the cultivation steps in a timely manner, resulting in problems such as large blood collection volume, complicated operation, and high cost
In order to solve the problem of low tumoricidal activity of single immune cells in adoptive cellular immunotherapy in the prior art, and to further improve the effect of immune cell therapy, it is necessary to combine two or more immune cells for treatment. The present invention can be very good achieve this purpose

Method used

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  • Method for simultaneously inducing and amplifying V alpha&lt;24+&gt;iNKT cells and CD&lt;3-&gt;CD&lt;56+&gt;NK cells
  • Method for simultaneously inducing and amplifying V alpha&lt;24+&gt;iNKT cells and CD&lt;3-&gt;CD&lt;56+&gt;NK cells
  • Method for simultaneously inducing and amplifying V alpha&lt;24+&gt;iNKT cells and CD&lt;3-&gt;CD&lt;56+&gt;NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Simultaneous induction and amplification of Vα 24+ iNKT cells and CD 3- cd 56+ NK cell method

[0050] 1. Induced amplification

[0051] On day 0, three cases of healthy human peripheral blood (100 mL per case) were collected, separated by gradient centrifugation with lymphocyte separation medium (trade name Lymphoprep TM, Axis-Shield Company, Norway), and the yellow layer on the upper layer of the centrifuge tube was collected as autologous Plasma, middle buffy coat was PBMC cells, the collected PBMC cells were washed twice with phosphate-buffered saline (PBS, GIBCO, U.S.A.), washed with AIM-V serum-free medium (trade name AIM-V Medium CTS , GIBCO, USA) to resuspend the cells, take 100L of the cell suspension and use the trypan blue exclusion method to detect the cell viability and count. According to the counting results, the obtained PBMC cells were adjusted with AIM-V serum-free medium to a concentration of 2×10 6 / mL, take out 1mL of cell suspension...

Embodiment 2

[0065] Example 2: Flow cytometry detection of Vα before and after amplification 24+ iNKT cells and CD 3- cd 56+ NK cell phenotype

[0066] The cryopreserved cells obtained in step 1 in Example 1 (that is, the 2×10 cells isolated on the 0th day of cryopreservation) 6 PBMC cells) are recovered, and the cells obtained after culturing for 15 days with the method of Example 1 are subjected to parallel flow detection operations, expressing CD 3- cd 56+ CD 3- cd 56+ NK cell percentage using anti-FITC Mouse Anti-Human CD 3 Detection antibody 20L (BDPharmingen, USA), PE Mouse Anti-Human CD 56 Detection antibody 20L (BD Pharmingen, USA) was double-labeled to express Va 24+ Va 24+ The percentage of iNKT cells was single-labeled with PE Mouse Anti-Human Invariant NKT cell antibody (BD Pharmingen, U.S.), and measured by a flow cytometer (Millipore guava easyCyte 6HT-2L, U.S.). figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , F...

Embodiment 3

[0069] Example 3: Cells highly express NKG2D after expansion

[0070] NKG2D is NK, T, CD 8+ Activating receptors of T cells, iNKT cells, macrophages, etc., NKG2D is involved in the recognition of virus-infected cells and the killing of tumor cells, and plays an important role in immune surveillance and immune killing.

[0071]In this example, NKG2D is used as a cell activation detection index. The recovered PBMC cells in Example 2 are subjected to parallel flow cytometric detection with the cells obtained after 15 days of culture in Example 1, and NKG2D antibody (BD Pharmingen, USA) is used for single-stage detection. Marker, measured by flow cytometer, measured values ​​are shown in Table 2, as can be seen from Table 2, the expression of NKG2D after sample amplification increased from 12.73%±3.71% before amplification to 80.25%±6.96% after amplification . Prove that the process of culturing induces Vα 24+ iNKT cells and CD 3- cd 56+ NKG2D, the activating receptor of NK c...

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Abstract

The invention discloses a method for simultaneously inducing and amplifying V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells. The method comprises the steps as follows: PBMC (peripheral blood mononuclear cells) are separated from peripheral blood, the concentration of the PBMC is adjusted to 2*10<6>/ml by a serum-free medium containing autologous plasma; an Anti-CD<16> antibody, -GalCer, IL-2, IL-18 and IL-21 are added, and then the mixture is transferred into a culture flask for culture; an Anti-CD3 antibody is added in a cell suspension in 24 hours; a serum-free medium containing IL-2, IL-18 and IL-21 is supplemented every two days according to the cell growth condition; the cell concentration is controlled to be 1.5*10<6>/ml; and after continuous culture is performed for 14-21 days, large quantities of high-purity V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells can be obtained simultaneously, and the total cell quantity can reach an effective value of the cell quantity required for adoptive cellular immunotherapy clinically for tumor. The method for simultaneous and efficient amplification of the V alpha<24+>iNKT cells and the CD<3->CD<56+>NK cells is simple, convenient and effective.

Description

technical field [0001] The invention belongs to the field of in vitro culture of immune cells, and relates to a method of simultaneously inducing and amplifying Vα by human peripheral blood mononuclear cells (PBMC) 24+ iNKT cells and CD 3- cd 56+ NK cell approach. Background technique [0002] Malignant tumor is a major disease that seriously threatens the health of our people. Traditional methods for treating malignant tumors include surgery, radiation therapy (abbreviated as radiotherapy) and chemotherapy (abbreviated as chemotherapy). Each of the three therapies has limitations: surgical treatment cannot remove the micrometastases of the tumor, and it is difficult to obtain a radical curative effect; radiotherapy has dose-dependent toxicity and cannot effectively remove potential metastases. In addition, some patients are resistant to radiotherapy due to radiation resistance. Insensitive, chemotherapy can easily cause systemic immunosuppression, and in some patients, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 邬冬云杨新娟廖娆君
Owner HRYZ (SHENZHEN) BIOTECH CO
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