Efficient tumor antigen loaded DC vaccine and method for induced proliferation of tumor antigen specific CTL by same

A tumor antigen, specific technology, applied in the fields of medicine and biology, to achieve efficient and specific killing effect

Active Publication Date: 2018-08-10
北京百益宁医学科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the polygenic changes of tumor cells, the high heterogeneity of tumor tissues, and the high degree of personalization of tumor patients, tumor-specific immunotherapy still faces great challenges

Method used

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  • Efficient tumor antigen loaded DC vaccine and method for induced proliferation of tumor antigen specific CTL by same
  • Efficient tumor antigen loaded DC vaccine and method for induced proliferation of tumor antigen specific CTL by same
  • Efficient tumor antigen loaded DC vaccine and method for induced proliferation of tumor antigen specific CTL by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of DC Vaccine Highly Efficiently Loaded with Tumor Antigens

[0049] 1. Tumor Cell Culture

[0050] Tumor cells come from the patient's own surgically resected tumor tissue or tumor tissue biopsy specimens, as well as tumor cell lines with the same pathological diagnostic characteristics as the patient. The cell line can be self-built in the laboratory, or can be purchased from China National Cell Collection Center or ATCC in the United States, and cultured in vitro according to the biological characteristics of the tumor cells. Usually use DMEM medium containing 10-15% fetal bovine serum at 37°C, 5% CO 2 Cultured in the incubator, subcultured and expanded.

[0051] 2. Induce cell apoptosis and express heat shock proteins

[0052] When the cells grow to the logarithmic phase and 80% of the bottom of the bottle is covered, the drug treatment is carried out, and arsenic trioxide (1-15umol / L can be added) is continued at 37 ° C, 5% CO 2 Incubate f...

Embodiment 2

[0071] Example 2 Preparation of DC Vaccine Highly Efficiently Loaded with Tumor Antigens

[0072] A method for preparing tumor antigens from tumor cells and exosomes in tumor cell culture fluid by separating tumor cells from the pleural and ascites of tumor patients, culturing and expanding them at one time.

[0073] 1. Collect 500-1000ml of pleural effusion or ascites from tumor patients under sterile conditions, centrifuge at 300g for 8 minutes, and discard the supernatant.

[0074] 2. Add physiological saline to 100ml to resuspend to make cell suspension.

[0075] 3. Double density centrifugation: from bottom to top: the first layer of 100% human lymphocyte separation liquid (density 1.077g / ml); the second layer of 75% lymphocyte separation liquid (density 1.077g / ml); the third layer cell suspension. Centrifugation: 700g, 20 minutes.

[0076] 4. Aspirate and collect the cells on the first interface and the second interface respectively, and wash with normal saline twice....

Embodiment 3

[0082] Example 3 Induction and expansion of tumor-specific CTL in vitro by tumor antigen-sensitized and activated DC

[0083] The DC vaccines prepared in Example 1 and Example 2 were selected respectively.

[0084] 1. On the day when the DC vaccine is harvested, draw 40-50 ml of heparin anticoagulated blood for the second time, and follow the operations of 1) and 2) in Step 6 of Example 1 to suck out the freshly separated non-adherent mononuclear cells, and use 5: The ratio of 1 was mixed with the prepared DC vaccine (DC vaccine prepared by referring to the method of Example 1 or Example 2), placed in a culture bottle containing serum-free CTL priming medium (BYN-PT331) overnight, and added anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, rh-IL-2, at 37°C, 5% CO 2 cultured in an incubator. Afterwards, according to the growth and expansion of the cells, an appropriate amount of serum-free expansion medium (BYN-PT332) containing rh-IL-2 was added in an ...

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Abstract

The invention provides an efficient tumor antigen loaded DC vaccine and a method for induced proliferation of tumor antigen specific CTL by the same. The DC vaccine is prepared by steps: (1) tumor antigen preparation; (2) acquisition, separation and efficient culture and proliferation of DC cells; (3) adoption of a tumor antigen and an activator for sensitization and activation of the DC cells; (4) DC cell surface PD-L1 molecule closing. The invention further provides a sequential blood drawing method for in-vitro induced proliferation of CTL by the DC vaccine and closing of surface PD-1 molecules of CTL subjected to in-vitro induced proliferation. Problems of low immunogenicity of existing tumor antigens, incompleteness of antigen targets, difficulty in acquisition of tumor antigens of patients and insufficiency in DC and CTL in-vitro proliferation are solved. The DC vaccine is effective in in-vitro and in-vivo induction of tumor antigen specific CTL and capable of efficiently generating specific killing effects on tumors without evident toxic and side effects, and a promising clinical application prospect is achieved.

Description

technical field [0001] The present invention relates to the fields of medicine and biotechnology, in particular to a low-initial amount of peripheral blood mononuclear cells for efficiently preparing DC vaccines loaded with autologous or other tumor antigens and a preparation method for inducing and amplifying tumor antigen-specific CTLs . Background technique [0002] Tumor is an inevitable product of human evolution, and it is also a major disease that threatens human health and life. At present, the incidence of cancer in my country is increasing year by year, but traditional surgery, chemotherapy, and radiotherapy all have relatively large limitations. Surgery can only remove the mass visible to the naked eye, but cannot completely remove tumor cells, and it does not change the unbalanced internal environment of tumor patients; chemotherapy and radiotherapy can kill some tumor cells, but a considerable part of solid tumors are not sensitive to radiotherapy and chemother...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00C12N5/0784
CPCA61K39/0011A61P35/00C12N5/0639C12N2501/51A61K2039/5154A61K2039/572
Inventor 徐迎新李力梁凯
Owner 北京百益宁医学科技有限责任公司
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