Anti-human-lung-cancer-stem-cell monoclonal antibody

A monoclonal antibody and hybridoma cell line technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problems of targeting tumor stem cells that have not been reported, and achieve the purpose of inhibiting the growth of lung cancer and inhibiting Effects on self-renewal, growth inhibition and recurrence

Inactive Publication Date: 2017-02-01
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Antibody drugs targeting tumor ste

Method used

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  • Anti-human-lung-cancer-stem-cell monoclonal antibody
  • Anti-human-lung-cancer-stem-cell monoclonal antibody
  • Anti-human-lung-cancer-stem-cell monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Mouse Monoclonal Antibody Binding to Human Lung Cancer Stem Cell Membrane Surface Antigen

[0030] A new method of primary culture combined with transient subculture was used to isolate continuous passage cell lines that can produce lung cancer stem cells from human lung cancer tissues, and to establish an in vitro serum-free suspension culture technology that can effectively enrich lung cancer stem cells. The obtained lung cancer stem cells were fixed with paraformaldehyde, and Balb / c mice were immunized until the anti-human lung cancer stem cell titer of immunized mouse serum reached 1:50,000, and the mouse spleen cells were fused with mouse myeloma cells SP2 / 0 form hybridomas. The hybridoma clones were prepared by the methylcellulose plate method, and after the clones grew, they were picked and cultured in 96-well plates. The culture supernatant of each hybridoma clone containing the monoclonal antibody secreted by this clone was collected f...

Embodiment 2

[0031] Example 2 Screening of mouse monoclonal antibodies that specifically recognize membrane surface antigens of human lung cancer stem cells

[0032] The A549 spherical cells cultured in serum-free suspension were gently blown into single cells, inoculated at 2000 / well in a 96-well plate, cultured in serum-free medium for 24 hours, washed with PBS containing 1% BSA, and placed in each well Add 100ul of the culture supernatant of a hybridoma clone and incubate at room temperature for 2 hours; after washing five times with 1% BSA in PBS, add biotin-labeled secondary antibody and react at room temperature for 30 minutes; wash again with 1% BSA in PBS for five times and label with Cy3 Avidin was reacted at room temperature for 30 minutes; after washing five times with 1% BSA in PBS, the reaction of the hybridoma monoclonal antibody supernatant in the monoclonal antibody library with lung cancer stem cells was judged by fluorescence microscopy, and mouse monoclonal antibodies cap...

Embodiment 3

[0033] Example 3 Identification of tumorigenicity of cells recognized by monoclonal antibody secreted by hybridoma cell LC-5A6

[0034] A549 cells were prepared into single cells by trypsin digestion, added LC-5A6 antibody (5.0 μg / ml), incubated at 37°C for 1 hour, washed twice with PBS, added Percp-goat anti-mouse IgG, and incubated at 37°C in the dark for 30 minutes , sorted LC-5A6 by flow cytometry + cells, LC-5A6 - Cells were inoculated subcutaneously on the right side of the back of each nude mouse, 6 mice in each group. Surface marker negative group and unsorted group were used as controls. The long and short diameters of subcutaneous transplanted tumors were measured twice a week, and the tumor volume was calculated using the formula V=(π / 6)×(long diameter×short diameter×short diameter). As shown in the results, inoculation of LC-5A6 + Cell 1×10 3 One can form a tumor at 1 month, and the rate of tumor formation at 2 months is 6 / 6, while LC-5A6 - Cell 1×10 5 All f...

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Abstract

The present invention discloses an anti-human-lung-cancer-stem-cell monoclonal antibody, and particularly relates to a new monoclonal antibody capable of specifically recognizing and inhibiting human lung cancer stem cells, a hybridoma cell line for producing the monoclonal antibody, and applications of the monoclonal antibody in preparation of drugs for treatment of human lung cancer recurrence. According to the present invention, the monoclonal antibody can significantly inhibit the self-renewal, the invasion and the migration of human lung cancer stem cells in vitro, can significantly inhibit the growth and the recurrence of human lung cancer transplantation tumors in vivo, and prolongs the survival time of tumor-bearing mice. The present invention further provides a monoclonal antibody production cell line mouse hybridoma cell line LC-5A6 having the preservation number of CGMCC No.10898.

Description

technical field [0001] The invention relates to an antibody against human lung cancer stem cells and the hybridoma cell line produced therefrom, as well as the application of the antibody in treating the growth and recurrence of lung cancer. Specifically, the present invention provides a monoclonal antibody that can bind to the natural membrane antigen of human lung cancer stem cells, and its application in preparing a medicament for treating human lung cancer. Background technique [0002] Lung cancer is a malignant tumor that seriously threatens human life and health. Its morbidity and mortality rank first among malignant tumors and are on the rise. Although the clinical diagnosis and treatment of lung cancer have made great progress compared with the past, surgical resection is ineffective for most multiple, metastatic or recurrent lung cancers, and a considerable number of patients are resistant to chemotherapeutic drugs, and radiotherapy is limited by important thoracic...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395A61P35/00C12N5/20C12R1/91
Inventor 孙力超杨治华冉宇靓
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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