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Gamma delta T-like alpha beta T cell and preparation method and application thereof

A cell and tumor tissue technology, applied in the field of biomedicine, can solve problems such as failure to achieve results

Active Publication Date: 2021-02-26
北京荟科柘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expanded γδT cells are difficult to meet the clinical needs. Even though the single γδT cells expanded by optimizing various induction conditions and expansion methods have been applied in the corresponding immune diseases and tumor diseases, they have not reached the target level after application. people's desired effect

Method used

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  • Gamma delta T-like alpha beta T cell and preparation method and application thereof
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  • Gamma delta T-like alpha beta T cell and preparation method and application thereof

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preparation example Construction

[0034] The present invention provides a method for preparing γδT-like αβT cells, comprising the following steps: using tumor tissue antigens to induce γδT cells, using the γδT cells as antigen-presenting cells to activate αβT cells, and obtaining bispecific γδT cells induced by tumor tissue antigens Cell stimulation activates γδT-like αβ T cells.

[0035] The present invention does not specifically limit the types of tumor tissue antigens, preferably including lung cancer, breast cancer, liver cancer, rectal cancer and gastric cancer, and the preparation methods of different types of tumor tissue antigens are the same. The preparation method of the tumor tissue antigen according to the present invention preferably comprises: (1) mixing and digesting the tumor tissue piece washed with PBS and the compound enzyme liquid; the volume of the compound enzyme liquid is twice the volume of the tumor tissue block ; Include trypsin, collagenase I and collagenase IV in the compound enzym...

Embodiment 1

[0062] 1. Preparation of tumor tissue antigens from lung cancer, breast cancer, liver cancer, rectal cancer and gastric cancer

[0063] 1.1 Place the fresh tumor tissue in a culture dish, add an appropriate amount of PBS, remove the area where other tissues retain tumor cells, and wash it with PBS.

[0064] 1.2 Put the tumor tissue into a new culture dish, add PBS, and use ophthalmic scissors to cut the tissue into about 1-2mm 3 sized pieces. Transfer to a 50ml centrifuge tube and rinse with PBS. After the tissue pieces sink automatically, remove the PBS.

[0065] 1.3 Add twice the volume of 0.25% trypsin: 2% collagenase I: 2% collagenase IV mixture, shake and digest in a 37°C water bath for 30-60 minutes.

[0066] 1.4 After the digestion of the tissue block is completed, suck out the upper liquid, filter it with a cell mesh with a pore size of 70 μm, and transfer it to a centrifuge tube containing an equal volume of complete medium to terminate the digestion.

[0067] 1.5 ...

Embodiment 2

[0093] Lung cancer antigens are extracted, loaded with γδT cells, and co-cultured with αβT cells, and the obtained γδT-like αβT cells have a specific killing effect on lung cancer cells.

[0094] 1. Count lung cancer cells, add CFSE to make the final concentration of CFSE reach 2μM, and treat at 37°C for 30min.

[0095] 2. After taking it out, centrifuge at 1000rpm for 5 minutes, you can faintly see the cells stained yellow, wash with PBS three times.

[0096] 3. Mixed culture with γδT-like αβT cells, 37°C, 5% CO 2 , saturated humidity, incubated for 24h.

[0097] 4. Add 1 μg / ml PI working solution, keep away from light for 30 minutes, and then flow up.

[0098] 5. CFSE, PI double-positive cells are the killed lung cancer cells, divided by the total number of lung cancer cells, is the killing rate.

[0099] According to the above method to test, the results are as follows Figure 4 As shown, when the effect-to-target ratio of γδT-like αβT cells to lung cancer cells is 1:1,...

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Abstract

The invention provides a gamma delta T-like alpha beta T cell and a preparation method and application thereof, and relates to the technical field of biological medicine. The gamma delta T cell is induced by using a tumor tissue extraction antigen, and the gamma delta T cell after antigen induction serves as an antigen presenting cell to activate an alpha beta T cell to obtain the gamma delta T-like alpha beta T cell. The number of CD3 and HLA-DR double-positive gamma delta T-like alpha beta T cells in the gamma delta T-like alpha beta T cell accounts for 70% or above of the total number of Tcells; and when the effector target ratio of the gamma delta T-like alpha beta T cell to tumor cells is 1:1, the killing activity of the gamma delta T-like alpha beta T cell is 73.85-78.21%.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a γδT-like αβ T cell and a preparation method and application thereof. Background technique [0002] Human T lymphocytes can be divided into αβT cells and γδT cells according to their surface T cell antigen receptors. Although γδT cells only account for 1-10% of T cells, they do not depend on MHC recognition, so they can resist tumor escape; low expression of CTLA4 and PD-1 molecules can resist tumor suppression; they can resist tumors with broad-spectrum killing function Edit; The tumor microenvironment is more tolerant to low glucose and hypoxia so that it can more effectively exert its killing function on tumors. Due to the extremely low content of γδT cells in peripheral blood, the clinical application of γδT cells as adoptive immune cells is greatly limited. At present, γδT cells are expanded from peripheral blood mononuclear cells, but the expansion factor...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K39/00A61P35/00
CPCC12N5/0636A61K39/0011A61P35/00C12N2500/84C12N2500/30C12N2501/2302C12N2502/1114C12N2501/2307C12N2501/24C12N2533/52C12N2533/50
Inventor 朱哲
Owner 北京荟科柘生物科技有限公司
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