Preparation method and application of efficient immunocompetent cell CpG-DCIK

A technology of immune activity and cells, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of difficult to obtain tumor antigens and patients' loss of surgical opportunities

Inactive Publication Date: 2015-05-27
SHANGHAI LIWO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technical solution disclosed in the above-mentioned patent document CN1431298A also has certain shortcomings and deficiencies: the preparation of tumor antigen-challenged DCIK cells must undergo surgery and other methods to obtain fresh tumor tissue specimens to extract antigens, and clinically, a considerable number of patients have lost the opportunity for surgery. And patients who have undergone radiotherapy and chemotherapy are also difficult to obtain tumor antigens
[0007] Therefore, it is urgent to improve the anti-tumor immune effector cells in the prior art in terms of proliferation performance, cytotoxic activity and specific recognition and attack activity of tumor cells, so as to develop a new anti-tumor immune effector cell.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of Efficient Immunocompetent Cell CpG-DCIK

[0035] Including the following steps:

[0036] Step (1) Preparation of Peripheral Blood Mononuclear Cells (PBMC)

[0037] Take 20-50ml of anticoagulant peripheral blood from tumor patients, dilute it with normal saline, and separate peripheral blood mononuclear cells (PBMC) with a lymphocyte separation medium with a specific gravity of 1.077. The separation uses a centrifuge with a horizontal rotor. The speed is 2000rpm, the centrifugation time is 20 minutes, and then washed with Hanks balance solution for 2 to 3 times before counting the cells under the microscope, and finally using the lymphocyte culture medium to completely adjust the cell density to 3 to 5x10 with F12 / DMEM1:1 -6 / ml of cell suspension;

[0038] Step (2) Separation of Peripheral Blood Lymphocytes (PBL) and Adherent Cells

[0039] Transfer the cell suspension described in step (1) into a 6-well plate, 3-5ml per well, and place at 37...

Embodiment 2

[0046] Example 2 Anti-tumor Application of Efficient Immunocompetent Cell CpG-DCIK

[0047] The right ear of nude mice was subcutaneously inoculated with human prostate cancer cells (Alva41) 1x10 -7 / only, tumor formation rate 100% after one week, perineal transfer 100% after 14 days, begin the CpG-DCIK cell suspension 2-3x10 that embodiment 1 obtains of continuous intraperitoneal inoculation -8 / only / time, once every other day, 4 times in total, until 35 days, the survival rate of the normal saline control group was 0% (0 / 4), the CIK cell therapy group was 20% (1 / 5), and the DCIK cell therapy group was 50% (3 / 6), the CpG-DCIK treatment group was 70% (6 / 8), and the tumor weight of the CpG-DCIK group was significantly smaller than the tumor weight of the CIK cell group, and the tumor weight difference with the DCIK group was not obvious, indicating that the CpG obtained in the above-mentioned embodiment 1 -DCIK has obvious anti-tumor immune activity.

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Abstract

The invention relates to a preparation method and an application of efficient immunocompetent cell CpG-DCIK. The preparation method comprises the following steps: inducing DC cell by use of CpG and cytokine, inducing homologous CIK cell by use of cytokine, and performing mixed culture of the DC cell and CIK cell to obtain a new immune effector cell population, namely oligonucleotide induced dendritic cell and cytokine induced killer cell co-culture cell which is named CpG-DCIK. According to the invention, the DC is induced by use of in-vitro CpG-ODN in combination with cytokine, and then the DC is co-cultured with homologous CIK to obtain an immune effector cell population with higher proliferation activity and cytotoxic activity, and an immune cell population with relatively high antitumor activity can still be induced and amplified for the cases in which a tumor antigen is hardly acquired since the operation opportunity is lost or the cancer is in the late stage and the like, thereby widening the application range of tumor resistance.

Description

technical field [0001] The invention belongs to the technical field of immune active cells, in particular to a preparation method and application of an anti-tumor immune active cell CpG-DCIK. Background technique [0002] Malignant tumors are currently one of the major diseases that endanger human health. In countries where infectious diseases are under control, cardiovascular and cerebrovascular diseases and malignant tumors have become the first and second causes of death, respectively. According to statistics in 2005, malignant tumors are the leading cause of death among urban residents in my country, and the second leading cause of death among rural residents. [0003] In the treatment of tumors, immunotherapy has become the most important and promising treatment method besides surgery, chemotherapy and radiotherapy. In the past 20 years, there have been great developments in both tumor-specific active immunotherapy and adoptive immunotherapy. In the adoptive immunothe...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/0783A61K35/17A61P35/00
Inventor 李丽
Owner SHANGHAI LIWO BIOTECH
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