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A monoclonal antibody against human liver cancer stem cells

A monoclonal antibody and liver cancer stem cell technology, which is applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problems of recurrence and metastasis of tumor patients, and achieve the purpose of inhibiting the growth of human liver cancer transplanted tumors in nude mice, inhibitory effect

Active Publication Date: 2020-01-10
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing treatment methods mainly kill sensitive tumor progeny cells, but have little effect on tumor stem cells, which eventually lead to recurrence and metastasis of tumor patients. Therefore, new therapeutic strategies targeting tumor stem cells are expected to reduce tumor metastasis. , recurrence and drug resistance, improving curative effect and reducing mortality will become the most effective means of tumor treatment

Method used

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  • A monoclonal antibody against human liver cancer stem cells
  • A monoclonal antibody against human liver cancer stem cells
  • A monoclonal antibody against human liver cancer stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of mouse monoclonal antibody against human liver cancer stem cells

[0029]A new method of primary culture combined with transient subculture was used to isolate a continuous passage cell line that can produce liver cancer stem cells from human liver cancer tissue, and to establish an in vitro serum-free suspension culture technology that can effectively enrich liver cancer stem cells. The liver cancer stem cells obtained were fixed with paraformaldehyde, and Balb / c mice were immunized until the titer of anti-human liver cancer stem cells in the 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 from the 96-wel...

Embodiment 2

[0030] Example 2 Screening of mouse monoclonal antibodies that specifically recognize human liver cancer stem cell membrane surface antigens

[0031] MHCC97L spherical cells cultured in serum-free suspension were blown gently into single cells, inoculated at 2000 / well in 96-well plates, 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 liver cancer stem cells was judged by fluorescence microscopy, and mouse monoclonal antibodies capable...

Embodiment 3

[0032] Example 3 Identification of the effect of the monoclonal antibody secreted by the hybridoma cell line HC-7B2 on the self-renewal of liver cancer stem cells

[0033] MHCC97L spherical cells cultured in serum-free suspension were gently blown into single cells, incubated with monoclonal antibody HC-7B2 (1 mg / ml) and incubated at 37°C for 2 hours, and seeded at 500 cells / well in low-adhesion cells. In a 24-well plate, use serum-free suspension culture containing 0.8% methylcellulose, and culture in a 5% CO2 incubator at 37°C for 14 days to calculate the sphere formation rate. See Table 1. The results showed that the number of spheres formed in the monoclonal antibody HC-7B2 treatment group was (69±17), while the number of spheres formed in the negative control group was (115±20), which was significantly higher than that of the antibody treatment group. The results showed Monoclonal antibody HC-7B2 can inhibit the self-renewal ability of liver cancer stem cells.

[0034] T...

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Abstract

The present invention discloses an anti-human-liver-cancer-stem-cell monoclonal antibody, and particularly relates to a new monoclonal antibody capable of inhibiting human liver cancer stem cells in a targeting manner, a hybridoma cell line secreting the monoclonal antibody, and applications of the monoclonal antibody in preparation of drugs for treatment of human liver cancer recurrence and drug resistance. According to the present invention, the monoclonal antibody can significantly inhibit the self-renewal, the invasion and the drug resistance of human liver cancer stem cells in vitro, can significantly inhibit the growth of human liver 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 HC-7B2 (with the preservation number of CGMCC No.10897).

Description

technical field [0001] The invention relates to an anti-human liver cancer stem cell monoclonal antibody and a hybridoma cell line produced therefrom, as well as the application of the antibody in treating recurrence and drug resistance of liver cancer. Background technique [0002] Liver cancer is one of the common malignant tumors in my country, the incidence rate accounts for 45% of the world, and the 5-year survival rate is only about 15-30%. The current clinical treatment methods for liver cancer, such as surgery, radiotherapy, chemotherapy and interventional therapy, have limited efficacy in the treatment of advanced liver cancer, and recurrence and drug resistance are the root causes of high mortality. [0003] Cancer stem cells are a small part of cancer cells with stem cell characteristics in tumor tissue, which have the ability of self-renewal and multidirectional differentiation, and play an important role in the metastasis, recurrence and drug resistance of malig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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