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Preparation method and application of tumor tissue complete antigen

A tumor tissue and whole antigen technology, which is applied in the field of preparation of tumor tissue whole antigen, can solve the problems of weak antigenicity, inability to obtain tumor antigens for patients, and inability to prepare tumor antigens

Inactive Publication Date: 2012-08-08
JIANGSU PROVINCIAL HOSPITAL OF TCM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the clinical application of DC vaccines is still limited by many aspects.
On the one hand, the immunogenicity of the vaccine is still not strong enough; on the other hand, most patients cannot obtain effective tumor antigens
[0008] Tumor-specific / related antigenic proteins, tumor-specific / related antigenic polypeptides have weak antigenicity, single target for activating immunity, poor vaccine immunogenicity, and most types of tumors have not been able to determine effective tumor-specific / related antigenic proteins. Sexual antigenic proteins and antigenic polypeptides are further restricted by HLA and can only be used for patients with specific HLA subtypes, so it is difficult to be widely used
Proteins and polypeptides containing various tumor antigens, such as tumor cells inactivated by radiation, tumor cell freeze-thaw supernatant or tumor cell membrane fragments, tumor cell subcellular components, etc., have strong antigenicity and are prepared after DC ingestion The tumor antigens carried by the vaccine have comprehensive targets and stimulate the body to produce comprehensive anti-tumor immunity. However, most types of tumors currently cannot obtain a sufficient number of tumor cells through in vitro culture methods, so it is difficult to obtain wide application
The more important problem is that cancer patients often receive DC vaccine immunotherapy after tumor recurrence, and at this time fresh tumor tissue has been lost, and tumor antigens cannot be prepared
[0009] The pathology department of the hospital has kept all the tumor tissues of all patients for a long time, but after formaldehyde fixation and paraffin embedding, it contains formaldehyde and paraffin, is a hard solid, and the antigen is denatured. Currently, it cannot be used to prepare effective tumor antigens, so it is also impossible to prepare DC. vaccine

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  • Preparation method and application of tumor tissue complete antigen

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preparation example Construction

[0071] The preparation method of the tumor vaccine provided by the present invention is suitable for the preparation of DC vaccines for various solid tumors, and the tumor is selected from lymphoma, myeloma, kidney cancer, prostate cancer, malignant melanoma, breast cancer, colon cancer, etc. Solid tumors; preferably kidney cancer, prostate cancer, malignant melanoma, lymphoma, and myeloma.

[0072] The present invention establishes a new preparation technology for DC vaccine. In a preferred example, the specific operation steps are as follows:

[0073] (1) Extract tumor antigen

[0074] 1. After formaldehyde fixation, the paraffin-embedded patient's tumor tissue is cut into 5-10μm thick slices;

[0075] 2. Mix the flakes and pure xylene to dissolve the paraffin;

[0076] 3. Centrifuge to remove xylene, add absolute ethanol, centrifuge several times to remove toxic organic solvents, and get the precipitate; then wash away the organic solvents with ethanol;

[0077] 4. Add an aqueous sod...

Embodiment 1

[0099] Preparation of lymphoma DC vaccine

[0100] (1) Extract tumor antigen

[0101] 1. After formaldehyde fixation, paraffin-embedded patient's lymphoma (diffuse large B-cell lymphoma) tumor tissue, cut 0.2g tumor tissue, cut into 5-10μm thick slices, and put it into a 15ml centrifuge tube;

[0102] 2. Add 10ml of 100% pure xylene, mix immediately, and incubate at 50°C for 5 minutes to dissolve the paraffin;

[0103] Centrifuge at 3.400g for 1 minute and discard xylene. Add 10ml of absolute ethanol, shake and mix;

[0104] 4. Centrifuge at 400g for 1 minute, discard ethanol, add 10ml absolute ethanol, shake and mix;

[0105] Centrifuge at 5.400g for 1 minute to remove residual ethanol as much as possible without destroying the precipitate;

[0106] 6. Add 20% Na 2 CO 3 10ml solution, put it in a 37℃ water bath for 1-6 hours;

[0107] 7. Centrifuge at 400g for 1 minute, discard all the liquid, add 10ml of water for injection, shake and mix well, and leave for 2 hours. Repeat this 3 time...

Embodiment 2

[0126] DC vaccine induces tumor-specific CTL in vitro

[0127] 1. Prepare the tumor antigen according to the method of Example 1.

[0128] 2. Prepare DC according to the method steps of Example 1.

[0129] 3. DC antigen shock: Prepare according to the relevant steps in Example 1 to obtain a DC vaccine.

[0130] 4. Grouping and in vitro cytotoxic T lymphocyte (CTL) culture:

[0131] Prepare patient's peripheral blood mononuclear cells, and use RPMI1640 medium containing 10% fetal calf serum (FCS) and recombinant human interleukin 2 (rh IL-2) (100U / ml) to make 5×10 5 / ml cell suspension, planted into 4 culture flasks, 20ml each bottle, and then add 1×10 to each flask 5 DC vaccine (ADC);

[0132] In the control group, DC (DC) and normal saline (NS) were added, and fresh complete medium and DC vaccine were supplemented on the 7th day of culture, and the cells were cultured to the 14th day.

[0133] 5. CTL detection: Taking patient tumor cells as target cells, LDH method detects CTL killing ac...

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Abstract

The invention discloses a preparation method and application of a tumor tissue complete antigen. The method comprises the following steps: (1) mixing paraffin embedded samples fixed by formalin (4-10% formaldehyde) of a bioptic tumor tissue with an organic solvent to dissolve the paraffin to obtain solution containing the tumor tissue; (2) centrifuging the solution containing the tumor tissue to obtain precipitate 1; (3) mixing the precipitate 1 with inorganic substance aqueous solution with pH being less than 7 for 0.5-12h and removing the liquid by centrifuging to obtain precipitate 2; (4) placing the precipitate 2 into water steam to inactivate the protease in the cytoplasm for 10-30min to obtain the tumor tissue which inactivates the protease in the cytoplasm; (5) homogenizing the tumor tissue obtained in the step (4) to obtain chyliform liquid; and (6) mixing the chyliform liquid and normal saline (or other solution) to obtain the tumor tissue complete antigen. The invention alsodiscloses a method for obtaining tumor vaccines by using the tumor tissue complete antigen to pulse the dendritic cell.

Description

Technical field [0001] The invention relates to the field of tumor immunotherapy, in particular to the preparation method and application of tumor tissue full antigen. Background technique [0002] At present, tumor vaccines are used to stimulate patients to generate anti-tumor immunity, that is, there are many methods for active immunotherapy of tumors, including tumor-specific / related antigen proteins, tumor-specific / related antigen polypeptides, plasmids expressing tumor antigens, Immune gene-modified tumor cells, dendritic cells (DCs) impacted by tumor antigens, etc. Among them, the DCs impacted by tumor antigens are the most promising tumor vaccines currently, namely dendritic cell-based vaccines. [0003] DC vaccine is a new tumor treatment vaccine developed in the past decade. The main mechanism is to immunize tumor patients with tumor antigen-carrying DCs and activate tumor-specific T cells in the body to induce specific and lasting tumors. Anti-tumor immune response to e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K14/47A61K39/00A61P35/00
Inventor 朱学军范振芳李晓惠孙雪梅
Owner JIANGSU PROVINCIAL HOSPITAL OF TCM
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