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

AI Technical Summary

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-targ

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

Example Embodiment

[0042] Example 1

[0043] Preparation of subsequent embodiments (cell culture, frozen memory and recovery)

[0044] First, cell culture

[0045] (1) All cells are placed at 37 ° C, 5% CO 2 The constant temperature incubator is cultured. (2) HCC827, HCC827 ER3: Cultivation using 10% RPMI-1640 fully medium. The HCC827 cells are converted, passaged or freezed in 2-3 days according to 1: 2-1: 3 pass. HCC827 ER3 cells are converted, passaged or freezing in 2-3 days in 1: 2. (3) A549: Cultivation using 10% DMEM / F12 fully medium. Press 1: 3-1: 6 for passage, 1-2 days to conduct liquid, pass or freeze. (4) 293T: Cultivation was carried out using 10% DMEM fully medium. Press 1: 3-1: 6 for passage, 1-2 days to conduct liquid, pass or freeze. 293T cells are half-pocket cells, the adhesive wall is not strong, and the culture process should avoid violently blow or violently shake, and the digestion time is less than 1 min. (5) 293F: Purification for AXL SCFV protein. Press 1: 2-1: 3 for pass...

Example Embodiment

[0050] Example 2

[0051] Expression of detecting lung cancer and human normal organs or tissue AXL

[0052] In order to verify AXL is an ideal target for treating lung cancer and to avoid potentially "derail" phenomena, this example detects 90 lung cancer (including 40 EGFR-TKi drug-resistant patients) tissue and 19 human body normal. AXL expression of organ or organization. At the same time, Western-blot was used to detect AXL at 8 on EGFR-TKI-resistant lung cancer and its corresponding cancerous lung tissue, thereby further acquisition of AXL specific expression relationship.

[0053] Specifically, the immunohard steps of this embodiment include: (1) baking sheet: placed tissue sections for 1-2 hours of oven baking sheet; (2) Death: Place the grill into xylene Death waxes 2 times, 10 minutes each time; (3) hydration: hydration in an orderly session of 100%, 95%, 75%, 50% ethanol, 5 minutes per cylinder; then wash 3 times in TBST 5 minutes each time; (4) antigen repair: placed t...

Example Embodiment

[0066] Example 3

[0067] Phenotypic detection of lung cancer cell line AXL

[0068] First, Western-blot detects the expression of lung cancer cell line AXL

[0069] 10cm Petri dish culture A549, HCC827 and HCC827 ER3 cells, the total protein was extracted from ice. The subsequent steps are described above, and the WB detection step is used in Example 1.

[0070] In this example and the following examples, human lung adenocarcinoma A549, HCC827 cells were purchased in ATCC. HCC827 ER3 is preserved by this topic laboratory, see Activation of the AXL Kinase Causes Resistance Toegfr-Targeted Therapy in Lung Cancer, HCC827 ER3 is an Erotinib drug resistant cell model for HCC827.

[0071] WB detection Figure 4 A shows that the HCC827 cell line does not express AXL. That is the same as the previous research group of the inventors, see Document Antability of the AXL Kinase Causes Resistance To Egfr-Targetedtherapy In Lung Cancer.

[0072] Second, flow cytometry detection lung cancer cell...

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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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