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Secretory target Lewis-Y CAR-T cell and preparation method and application thereof

A lewis-y-car, secreted technology, applied in the field of CAR-T cells, can solve the problem of limited ability to recognize and kill tumor cells, and achieve the effect of enhancing anti-tumor ability, enhancing the effect of killing tumor cells, and enhancing immune memory

Active Publication Date: 2019-04-23
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the CAR structure used to treat CD174-positive tumors is the second-generation CAR structure. Although CAR-T cells with this structure have strong antigen recognition and binding capabilities, due to the complex tumor microenvironment of solid tumors, CAR-T cells cannot Good infiltration into tumor tissue, and the function of CAR-T cells will be inhibited due to the combination with PD-L1 molecules on the surface of tumor cells. Therefore, the ability of the second-generation CAR-T to recognize and kill tumor cells is limited

Method used

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  • Secretory target Lewis-Y CAR-T cell and preparation method and application thereof
  • Secretory target Lewis-Y CAR-T cell and preparation method and application thereof
  • Secretory target Lewis-Y CAR-T cell and preparation method and application thereof

Examples

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Embodiment 1

[0057] Example 1: Preparation of CAR-T cells expressing Lewis-Y-CAR while secreting IL12.

[0058] The preparation method of CAR-T cells expressing Lewis-Y-CAR and secreting IL12 in the present invention is as follows:

[0059] 1. Isolate PBMCs from peripheral blood provided by donors.

[0060] 1. Collect 80-100mL of human peripheral blood with anticoagulant tube or blood collection bag (shake while collecting to make the peripheral blood and anticoagulant fully mixed), and separate PBMC in a sterile cell preparation workshop. Transfer the peripheral blood to a 50ml centrifuge tube, centrifuge at 800g at room temperature for 15 minutes, absorb the upper layer of yellow autologous plasma into a new 50ml centrifuge tube, inactivate at 56°C for 30 minutes, and store it at -20°C for later use; add to the lower cell layer Equal volume of PBS, mix up and down;

[0061] 2. Draw 15ml of lymphocyte separation medium (Ficoll-PaquePlus, GE) at the bottom of a new 50ml centrifuge tube, ...

Embodiment 2

[0113] Example 2: Preparation of CAR-T cells expressing Lewis-Y-CAR and secreting PD-1 antibody.

[0114] The preparation method of CAR-T cells expressing Lewis-Y-CAR and secreting PD-1 antibody in the present invention is as follows:

[0115] 1. Isolate PBMCs from peripheral blood provided by donors.

[0116] 1. Collect 80-100mL of human peripheral blood with anticoagulant tube or blood collection bag (shake while collecting to make the peripheral blood and anticoagulant fully mixed), and separate PBMC in a sterile cell preparation workshop. Transfer the peripheral blood to a 50ml centrifuge tube, centrifuge at 800g at room temperature for 15 minutes, absorb the upper layer of yellow autologous plasma into a new 50ml centrifuge tube, inactivate at 56°C for 30 minutes, and store it at -20°C for later use; add to the lower cell layer Equal volume of PBS, mix up and down;

[0117] 2. Draw 15ml of lymphocyte separation medium (Ficoll-Paque Plus, GE) at the bottom of a new 50ml ce...

Embodiment 3

[0168] Example 3: Targeted Lewis-Y-CAR-T cells that can secrete IL12 or PD-1 antibodies are used for immunotherapy.

[0169] 1. Use immunohistochemistry to detect whether the tumor tissue cells of tumor patients highly express Lewis-Y. Lewis-Y is highly expressed in various tumors, such as lung cancer and gastric cancer. Any tumor patient with high expression of Lewis-Y antigen can be used Treatment with targeted Lewis-Y-CAR-T cells that secrete IL12 or PD-1 antibodies.

[0170] 2. Collect peripheral blood from the patient and isolate T cells.

[0171] 3. Preparation of CAR-T cells. Prepare CAR-T cells in a GMP workshop. After the isolated T cells were activated with CD3 / CD28 beads, T cells were transduced with lentivirus according to MOI=3-10, and CAR-T cells were expanded in vitro.

[0172] 4. After the CAR-T cells are cultured in vitro to meet the number of therapeutic cells, some T cells are extracted for sterility testing and T cell phenotype testing, and can be used fo...

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Abstract

The invention discloses a secretory target Lewis-Y CAR-T cell, which comprises a CAR-T cell which targets Lewis-Y and secretes IL12 cytokine; a CAR-T cell which targets Lewis-Y and secretes PD-1 antibodies. The invention further discloses a preparation method of the CAR-T cell of the secretory target Lewis-Y and an application of the CAR-T cell in preparing an anti-tumor cell therapy medicament. The PD-1 antibody secreted by the CAR-T cell is specifically combined with the PD-1 on the surface of the T cell, thereby blocking the inhibition reaction of the PD-1 / PD-L1 signal on the activity of the T cell, so that the effective inhibition of growth of tumor cells for a long time by the CAR-T cell is facilitated; the IL12 cell factor secreted by the CAR-T cell can act on immunosuppressive cellsin tumor inhibitory immune microenvironment, so that the immunosuppressive capacity of the immunosuppressive cells is reduced, and the killing effect of the CAR-T cell is further enhanced.

Description

technical field [0001] The present invention relates to the fields of immunology, cell biology and molecular biology, and in particular to a CAR-T cell that can secrete IL12 or PD-1 antibody and targets Lewis-Y. The carrier prepares the method for preparing the cell and the application of the cell. technical background [0002] "Cancer Statistics in China, 2015" published in 2016 shows that in 2015, China is expected to have 4.292 million new cancer cases and 2.814 million deaths. Among them, the cancers with the highest mortality rate are: lung cancer, bronchus cancer, gastric cancer, liver cancer, esophageal cancer and colorectal cancer, accounting for three quarters of all cancer deaths. According to the latest data released by WHO, cancer deaths account for about 23% in my country, second only to cardiovascular diseases. With the intensification of population aging in my country, environmental pollution brought about by industrialization, and changes in living habits b...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N15/86C07K14/5434C07K16/2818C07K16/2896C12N2510/02C12N2740/15043C07K2319/33
Inventor 李甲璐尚小云许先进王丹马丽马少文
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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