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CAR-T cell targeting AXL and preparation method and application thereof

An EGFR-TKI, cell technology, applied in the field of immunology, can solve the problems such as the treatment of solid tumors is not as effective as hematological tumors, difficult to find tumor targets, off-target effects, etc., to improve the scope of tumor treatment, overcome drug resistance, and inhibit tumors. effect of growth

Pending Publication Date: 2022-02-22
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when using CAR-T therapy to treat solid tumors, there are many problems, including: it is difficult to find a tumor target as specific as CD19 in the treatment of hematological tumors, and it is prone to off-target effects and side effects; the heterogeneity of solid tumors is easy Causes conditions such as drug resistance due to antigen loss; its effect on solid tumors is significantly less than that on hematologic malignancies

Method used

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  • CAR-T cell targeting AXL and preparation method and application thereof
  • CAR-T cell targeting AXL and preparation method and application thereof
  • CAR-T cell targeting AXL and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparatory work (cell culture, cryopreservation and recovery) of follow-up embodiment

[0044] 1. Cell culture

[0045] (1) All cells were placed at 37°C, 5% CO 2 Constant temperature incubator for cultivation. (2) HCC827, HCC827 ER3: cultured with 10% RPMI-1640 complete medium. HCC827 cells were passaged according to 1:2-1:3, and the medium was changed, passaged or frozen in 2-3 days. HCC827 ER3 cells were passaged at a ratio of 1:2, and the medium was changed, passaged or frozen for 2-3 days. (3) A549: cultured with 10% DMEM / F12 complete medium. Subculture according to 1:3-1:6, 1-2 days for medium change, passage or freezing. (4) 293T: cultured with 10% DMEM complete medium. Subculture according to 1:3-1:6, 1-2 days for medium change, passage or freezing. 293T cells are semi-adherent cells, and the adherence is not firm. During the culture process, violent blowing or shaking should be avoided, and the digestion time should be less than 1min. (5) 293F: use...

Embodiment 2

[0051] Detection of AXL expression in lung cancer and normal human organs or tissues

[0052] In order to verify that AXL is an ideal target for the treatment of lung cancer and avoid potential "off-target" phenomena, this example detected 90 cases of lung cancer (including 40 cases of EGFR-TKI drug-resistant patients) tissues and 19 human normal tissues by immunohistochemical method. AXL expression in organs or tissues. At the same time, Western-Blot was also used to detect the expression of AXL in 8 pairs of EGFR-TKI resistant lung cancer and its corresponding paracancerous lung tissues, so as to further obtain the specific expression relationship of AXL.

[0053] Specifically, the immunohistochemical operation steps described in this example include: (1) Baking slices: place the tissue slices in an oven at 65°C for 1-2 hours; (2) Dewaxing: put the grill in xylene Dewaxing 2 times, 10 minutes each time; (3) Hydration: orderly hydration in 100%, 95%, 75%, 50% ethanol respect...

Embodiment 3

[0067] Phenotype detection of lung cancer cell line AXL

[0068] 1. Western-Blot detection of AXL expression in lung cancer cell lines

[0069] A549, HCC827 and HCC827 ER3 cells were cultured in a 10 cm culture dish, and the total protein was extracted on ice after overnight without serum. See the above for the subsequent steps, using the WB detection step in Example 1.

[0070] In this example and the following examples, human lung adenocarcinoma A549 and HCC827 cells were purchased from ATCC. HCC827 ER3 is preserved by the laboratory of our research group, please refer to Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer, HCC827 ER3 is an Erotinib-resistant cell model of HCC827.

[0071] WB detection such as Figure 4 In A, the HCC827 cell line does not express AXL. It is the same as the previous research of the inventor's research group, see the document Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung ...

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Abstract

The invention discloses an application of a CAR-T cell targeting AXL, a third generation of CAR-T cell targeting AXL is prepared by using scFv protein targeting AXL, and the CAR-T cell is applied to tumor treatment, so that the problem that a tumor specific target is difficult to find when CAR-T is used for treating solid tumors is solved, target missing is effectively avoided, the probability of side effects is reduced, and the solid tumor treatment effect is improved. Based on high expression of the AXL in lung cancer, the AXL has a specific killing effect on lung cancer tissues with positive expression of the AXL, so that the treatment on the lung cancer is realized; especially, the cell acts on lung cancer cells which are resistant to EGFR-TKI due to up-regulation of AXL, and can significantly improve the treatment effect of lung cancer and overcome the poor treatment effect caused by drug resistance in cooperation with EGFR-TKI drugs in treatment of lung cancer.

Description

technical field [0001] The present invention relates to the field of immunology, and more specifically, to a CAR-T cell targeting AXL and its preparation method and application. Background technique [0002] Lung cancer is one of the most common malignant tumors worldwide. According to the statistics of the National Central Cancer Registry of China (NCCR), the incidence and mortality of lung cancer rank first in my country. In recent years, although the treatment of lung cancer has made great progress, such as surgery, radiotherapy, chemotherapy and targeted therapy. However, since many patients have local or extensive metastases when lung cancer is discovered, the overall curative effect of lung cancer is still very limited, and the 5-year survival rate is only about 15%. Therefore, a new treatment strategy or drug is urgently needed to improve the treatment effect and survival rate of lung cancer patients. [0003] CAR-T therapy (Chimeric Antigen Receptor T-Cell Immunot...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K39/00A61P35/00
CPCC12N5/0636C12N15/86C07K14/7051C07K16/40A61K39/001156A61P35/00C12N2740/15043C12N2510/00C07K2319/03A61K2039/5158
Inventor 张振峰曹碧辉刘曼婷杨丽丽陈德基练辉周玉波彭韧李俊平冯云飞王露陈小佩田宇吴镜强刘敏刘玲
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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