Preparation and applications of liver-cancer-specific CTL cells induced by new enhanced antigen combined polypeptide

A specific technology for liver cancer cells, applied in the direction of fusion polypeptides, tumor-specific antigens, hybrid peptides, etc., can solve the problems of many side reactions, short antibody half-life, high treatment cost, etc., to achieve specificity enhancement and enhancement effect. , the effect of enhancing the killing effect

Inactive Publication Date: 2017-04-26
闾军 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, more clinical applications are specific antibodies against tumor targets. The half-life of antibodies in the body is short, and repeated administration is required at intervals. The treatment cost is expensive and there are many side effects.

Method used

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  • Preparation and applications of liver-cancer-specific CTL cells induced by new enhanced antigen combined polypeptide
  • Preparation and applications of liver-cancer-specific CTL cells induced by new enhanced antigen combined polypeptide
  • Preparation and applications of liver-cancer-specific CTL cells induced by new enhanced antigen combined polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Synthesis of Liver Cancer-specific Combined Polypeptide

[0045] HLA molecules generally bind to polypeptides of specific length and affinity, and the amino acid residues at specific positions determine the affinity of polypeptides recognized by TCR. We used the SYFPEITHI and BIMAS databases to predict the binding ability of GPC3, AFP, NY-ESO-1 polypeptides and MHC-I molecules, and then compared and scored and screened. We obtained the new sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 of GPC3, AFP, NY-ESO-1 with high affinity for HLA molecules. After reviewing a large number of relevant literature and patent reports, the above sequences have not been published in related research.

[0046] The amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and the classic antigen peptide group (sequence has been reported) SEQ ID NO: 5 obtained by screening were entrusted to Beijing Baisheng Gene Technology Co., Ltd. adopts TETRAS TM ...

Embodiment 2

[0047] Example 2 Peripheral Blood Mononuclear Cells (PBMC) Extraction

[0048] (1) Separate 20ml of anticoagulated venous blood from patients with HLA-A*0201 positive liver cancer in the morning. Add 15ml Lymphoprep Separation Solution to a 50ml centrifuge tube.

[0049] (2) Slowly add an equal volume of 0.9% sterile normal saline to the anticoagulated venous blood and gently invert three times to mix well.

[0050] (3) Use a 2ml sterile dropper to slowly add the diluted blood to the surface of the lymphocyte separation medium, and do not shake or turn it upside down after all is transferred.

[0051] (4) Balance the centrifuge tube and centrifuge at room temperature for 30 min at 1700 rpm in a horizontal centrifuge (horizontal rotor), ace / brake: 0 / 0.

[0052] (5) Aspirate excess plasma from the uppermost layer. Gently aspirate the lymphocyte layer with a 2ml sterile pipette, transfer to a new 50ml centrifuge tube, and aspirate the lymphocyte layer in all tubes into the sam...

Embodiment 3

[0057] Example 3 Preparation of Antigen Presenting Cells

[0058] (1) Resuspend PBMC obtained by density gradient centrifugation in AIM V medium, add to cell culture dish, 37°C, 5% CO 2 nourish.

[0059] (2) After 3 hours, take out the culture dish, shake it gently, and suck out the suspended cells.

[0060] (3) AIM V medium containing 500 IU / ml rhIL-4 and 500 IU / ml rhGM-CSF was added to the culture dish.

[0061] (4) On the 3rd day and the 5th day after culture, the medium was changed in half, and fresh medium containing 500 IU / ml of rhIL-4 and 500 IU / ml of rhGM-CSFAIM V medium was added.

[0062] (5) On the 6th day of culture, a culture medium with final concentrations of 10 ng / ml rhTNF-α, 300 IU / ml rhIL-2, and 10 ng / ml rhIL-33 was added.

[0063] (6) Mature dendritic cells were harvested on day 7.

[0064] (7) Mature dendritic cells were harvested by centrifugation, resuspended in AIM V medium, added with a final concentration of 10 μg / ml amino acid sequence of SEQ ID N...

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Abstract

The invention provides preparation and applications of liver-cancer-specific CTL cells induced by a new enhanced GPC3-AFP-NY-ESO-1 combined polypeptide. According to the present invention, new liver cancer cell surface antigen polypeptides are screened from GPC3, AFP and NY-ESO-1 are subjected to sequential fragment linking, and the newly screened GPC3 peptide, the newly screened AFP peptide and the newly screened NY-ESO-1 peptide are sequentially linked to form the CTL epitope peptide SEQ ID NO:4; and the new method and means for efficiently and specifically killing liver cancer tumor cells is established, the key problems of low tumor killing efficiency, long culture time and the like are overcome with the obtained liver-cancer-specific CTL cells, the liver cancer immunotherapy treatment effect can be specifically enhanced, and the tumor treatment application prospect is provided.

Description

[0001] Technical field: [0002] The invention belongs to the field of immune cell preparation, and specifically relates to a preparation method and application of a novel enhanced GPC3-AFP-NY-ESO-1 combined with polypeptide to induce liver cancer-specific CTL cells. [0003] Background technique: [0004] my country is a country with a high incidence of primary hepatocellular carcinoma in the world. There are about 1 million new cases of liver cancer every year in the world, and my country accounts for 55%. Statistics from my country's Cancer Registry Network in 2015 show that among males before the age of 60, liver cancer accounted for the highest proportion of deaths. In recent years, although there have been some breakthroughs in treatment, such as surgery, interventional therapy, and transplantation, the rapid progression and metastasis of liver cancer are still key problems to be overcome. Tumor immunotherapy has become an important means and method for treating and elim...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/0783A61K35/17A61K39/00A61P35/00
CPCA61K35/17A61K39/0011C07K14/4748C07K2319/00C12N5/0638C12N5/0639C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2307C12N2501/2315C12N2501/2321C12N2501/2323C12N2501/2333C12N2501/25C12N2501/515C12N2501/998C12N2502/1114
Inventor 闾军陈辉孙文峰
Owner 闾军
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