Isolated culture method of tumor-specific T cells and product obtained by isolated culture method

A tumor-specific, separation and culture technology, applied in the field of separation and culture methods and products obtained therefrom, to achieve the effects of convenient blood collection, short time required and high tumor specificity

Active Publication Date: 2020-11-20
HENAN CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method can only get CD4 + T lymphocytes

Method used

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  • Isolated culture method of tumor-specific T cells and product obtained by isolated culture method
  • Isolated culture method of tumor-specific T cells and product obtained by isolated culture method
  • Isolated culture method of tumor-specific T cells and product obtained by isolated culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Effect of Factor Types in the Culture Medium in Example 1 on the Effector Memory T Lymphocyte Ratio in Cell Final Products

[0057] This example explores the effects of five different culture methods on effector memory T cells (specific phenotype: CD3) in the final cell product. + CD8 + CD45RA - CCR7 - and CD3 + CD4 + CD45RA - CCR7 - ) ratio, its specific operation includes the following steps:

[0058] (1) Take the peripheral blood of kidney cancer patients who have been treated with PD-1 antibody within 3 weeks;

[0059] (2) The peripheral blood obtained in step (1) is separated by the Ficoll density gradient method to obtain mononuclear cells;

[0060] (3) The mononuclear cells obtained by step (2) are separated and used at a concentration of 10 7 Mononuclear cells were co-incubated with 2.5 μg of biotinylated anti-human IgG4 antibody for 20 minutes at 25°C, then added anti-biotin antibody-coated magnetic beads and incubated for 10 minutes at 25°C. Magnetic ...

Embodiment 2

[0063] Example 2 Effects of Two Different PD-1 Antibody Pretreatment Methods on the Proliferative Ability of PD-1 Positive Cells

[0064] This example explores the effects of two different PD-1 antibody pretreatment methods on the expansion speed of isolated tumor-specific T cells. In this example, two experiments were carried out on samples, one of which was peripheral blood from patients with cervical cancer. (sample 1), and the second is the peripheral blood (sample 2) of a patient with malignant melanoma. The specific operation includes the following steps:

[0065] (1) Collect peripheral blood from patients with cervical cancer and malignant melanoma before and after PD-1 antibody drug treatment (within 3 weeks). Add PD-1 antibody to the peripheral blood of patients who have not been treated with PD-1 antibody drug to make the final concentration 0.5 μg / mL, and incubate at 4°C for 0.5h, as pretreatment group 1; The patient's peripheral blood after drug treatment is direc...

Embodiment 3

[0069] Example 3 Evaluation Test of the Tumor Specificity of the Isolated T Cells (The Sample Is the Peripheral Blood of Renal Cancer Patients)

[0070] This example evaluates the ability of isolated T cells to recognize autologous tumor cells, using IFN-γ-secreting cells to account for CD8 + and CD4 + The proportion of cells (ie, CD8 + IFN-γ + %, CD4 + IFN-γ + %) for characterization, the higher the ratio, the higher the tumor specificity of T cells.

[0071] The specific operation includes the following steps:

[0072] (1) The test is divided into three groups, which are unsorted group, PD-1 negative group and PD-1 positive group;

[0073] (2) Take peripheral blood from kidney cancer patients, add PD-1 antibody to make the final concentration 0.5 μg / mL and incubate (pretreatment);

[0074] (3) The peripheral blood obtained in step (2) is separated by the Ficoll density gradient method to obtain mononuclear cells;

[0075] (4) The mononuclear cells obtained by step (3) ...

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Abstract

The invention relates to an isolated culture method of tumor-specific T cells and a product obtained by the isolated culture method. The isolated culture method comprises the following steps: firstly,pretreating peripheral blood of a tumor patient, then separating mononuclear cells in the peripheral blood, then separating PD-1 positive mononuclear cells, and finally amplifying the PD-1 positive mononuclear cells to obtain the tumor-specific T cells. According to the method for isolated culture of the tumor-specific T cells, the initial material is peripheral blood of the tumor patient, tumortissue or malignant pleural effusion of the patient does not need to be used as the material, materials are more convenient to obtain, blood sampling can be conveniently conducted on almost all tumorpatients, and large trauma is avoided; the finally obtained T cells are high in tumor specificity and contain CD4 positive T cells and CD8 positive T cells at the same time; the method is simple in operation process and short in required time.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for separating and culturing tumor-specific T cells and products obtained therefrom. Background technique [0002] Malignant tumors seriously endanger human life and health, and its mortality rate ranks first among various diseases. The three existing conventional treatment methods for tumors (i.e. surgery, radiotherapy and chemotherapy) have made significant contributions to improving the survival rate of cancer patients in the past few decades, but they have not been able to solve the problem of tumor metastasis and recurrence. However, the survival rate of cancer patients has not improved significantly in recent years. The three conventional treatments have encountered curative effect bottlenecks, and there is an urgent need to find new methods that can break through the bottleneck of tumor therapeutic efficacy. [0003] Tumor-specific T lymphocytes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2509/00C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/2321C12N2501/727C12N2533/52C12N2501/515
Inventor 李铁鹏高全立王瑶吕璐璐
Owner HENAN CANCER HOSPITAL
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