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Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine

A kit and a new type of technology are applied in the field of preparing high-efficiency new autologous DC vaccines, which can solve the problem of difficulty in exerting anti-tumor effects, and achieve the enhancement of pDC immune stimulation function, specific immune killing and anti-tumor, and DC migration ability. Effect

Active Publication Date: 2016-05-04
普济生物科技发展(山东)有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, DCs in tumor patients are affected by various immunosuppressive factors, and it is difficult to exert their anti-tumor effect.

Method used

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  • Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine
  • Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine
  • Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Culture of DC by GM-CSF+IL-4+FLT3-L+ATRA cytokine combination

[0040] Prepare a novel autologous DC vaccine with high efficiency as follows, including the following steps:

[0041] 1) Preparation of mononuclear cells: extract 50-100ml of peripheral blood from tumor patients under sterile conditions or about 80-100ml of peripheral blood mononuclear cells from patients with apheresis, transfer them into a 50ml centrifuge tube, add an equal volume of normal saline to dilute, Then transfer it to the top of the lymphocyte separation medium according to the volume of 2:1 or 1:1, 2000 rpm, slowly rise and fall (rise 1 fall 0), centrifuge for 20-30 minutes, absorb the buffy coat layer, which is a single nucleus Cells were washed twice with normal saline or Hanks solution, centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was PBMCs;

[0042] 2) Preparation of DC precursor cells: PBMCs in a culture flask, 37°C, 5% CO 2 Incubate for 1-2 ...

Embodiment 2

[0051] The semi-adherent PBMCs in step 2) of Example 1 were cultured with 500-1000U / mlGM-CSF+500-1000U / mlIL-4+50-200ng / mlFLT3-L+1-5uMATRA+1-5% autologous plasma Immature DCs were obtained after 4-5 days in the culture medium, and mature DCs were obtained as follows:

[0052] 1) Loading the obtained immature cells with 100ug / ml antigen for 24h;

[0053] 2) Divide the cells in the above step 1) into 2 groups:

[0054] Group 1: stimulated with 500-2000U / ml TNF-α for 24h;

[0055] Group 2: Stimulated with TLRs agonists mixed with 0.1-5KE / mlOK432+1-5uMCPGODN2336 for 24h;

[0056] Group 3: stimulated with TLRs agonists mixed with 0.1-5KE / mlOK432+1-5uMCPGODN2006 for 24h;

[0057] Group 4: Stimulated with TLRs agonists mixed with 0.1-5KE / mlOK432+1-5uMCPGODN2395 for 24h;

[0058] Group 5: (DC vaccine of the present invention) stimulated with TLRs agonists mixed with 0.1-5KE / mlOK432+1-5uMCPGODN21798 for 24h;

[0059] The DC maturation markers of each group in step 2) were detected ...

Embodiment 3

[0067] The DC vaccine obtained by the OK432+CPGODN21798 combined stimulation method with the strongest ability to secrete IL-12p70 and the lowest expression of PD-L1 was selected and compared with the DC vaccine obtained by the TNF-α method to detect the migration ability of mature DC and the ability to induce T cell proliferation and activation.

[0068] The semi-adherent PBMCs in the step 2) of embodiment one are divided into 2 groups:

[0069] Group 1 (traditional DC vaccine): After cultured with GM-CSF+IL-4 for 4-5 days, the antigen was loaded for 24 hours, and TNF-α stimulated maturation for 24 hours;

[0070]Group 2 (DC vaccine of the present invention, the same DC vaccine as Group 5 in Example 2): after culturing for 4-5 days with GM-CSF+IL-4+FLT3-L+ATRA, 100ug / ml antigen loading for 24h, and then Stimulate maturation with OK432+CPGODN21798;

[0071] The prepared DC vaccine was mixed with T cells and cultured for 24 hours.

[0072] WesternBlot detects the expression l...

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Abstract

The invention discloses a kit, method and application of a high-efficiency novel DC autovaccine, and belongs to the technical field of biology. The kit comprises a cytokine combination of GM-CSF, IL-4, FLT3-L and ATRA. According to the method, an existing GM-CSF+IL-4 cultivation technique is improved, is combined with FLT3-L to promote generation of pDC, mononuclear cells are increased to convert DC, generation of MDSC is inhibited by utilizing all-transretinoic acid (ATRA) to eliminate the negative adjustment effect of MDSC in preparation of DC vaccines, and an OK432 and CPH ODN co-stimulation mode is adopted to effectively promote mature of mDC and pDC and enhance the DC immunostimulation function.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit, method and application for preparing a highly efficient novel autologous DC vaccine. Background technique [0002] According to the World Cancer Report 2014 published by the International Agency for Research on Cancer under the World Health Organization, the number of cancer patients worldwide is increasing at an alarming rate. In 2012, there were an estimated 14 million new cases of cancer worldwide, and this number is expected to rise to 22 million within 20 years. Cancer deaths will also soar from 8.2 million to 13 million a year over the same period. The current main treatment methods for malignant tumors include surgery, radiotherapy, chemotherapy and targeted therapy. However, even with the adoption of a multidisciplinary treatment model, the mortality rate of cancer remains high. Novel cancer treatments are urgently needed. [0003] "SCIENCE" magazine rated tumor im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C12N5/0784A61P35/00
CPCA61K39/0011A61K2039/5154C12N5/0639C12N2501/22C12N2501/2304C12N2506/11
Inventor 罗昀
Owner 普济生物科技发展(山东)有限责任公司
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