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Preparation method and application of tumor tissue complete antigen

A tumor tissue and pan-antigen technology, applied in the field of tumor tissue pan-antigen preparation, can solve the problems of insufficient vaccine immunogenicity, inability to obtain a sufficient number of tumor cells, and single activation of immune targets.

Inactive Publication Date: 2010-06-09
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 applicable to 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, or colon cancer, etc. Solid tumors; preferably kidney cancer, prostate cancer, malignant melanoma, lymphoma, myeloma.

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

[0073] (1) Extraction of tumor antigens

[0074] 1. Formaldehyde-fixed and paraffin-embedded patient tumor tissue, cut into 5-10 μm thick slices;

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

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

[0077] 4. Add an aqueo...

Embodiment 1

[0099] Preparation of Lymphoma DC Vaccine

[0100] (1) Extraction of tumor antigens

[0101] 1. For the lymphoma (diffuse large B-cell lymphoma) tumor tissue of the patient who was fixed in formaldehyde and embedded in paraffin, cut 0.2g of tumor tissue into 5-10μm thick slices and put them 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] 3. 400g, centrifuge for 1 minute, discard xylene. Add 10ml of absolute ethanol, shake and mix;

[0104] 4. 400g, centrifuge for 1 minute, discard the ethanol, then add 10ml of absolute ethanol, shake and mix;

[0105] 5. 400g, centrifuge for 1 minute, remove residual ethanol as much as possible without destroying the precipitate;

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

[0107] 7. Centrifuge at 400g for 1 minute, discard all the liquid, then add 10ml of water for injection, vortex and mi...

Embodiment 2

[0126] Induction of tumor-specific CTL by DC vaccine 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 challenge: Prepare according to the relevant steps of Example 1 to obtain DC vaccine.

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

[0131] The peripheral blood mononuclear cells of patients were prepared, and 5×10 5 / ml of cell suspension, seeded into 4 culture flasks, 20ml in each flask, and then added 1×10 5 DC vaccine (ADC);

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

[0133] 5. CTL detection: the patient's tumor cells were used as target cells, and the killing activity of CTL in vitro was detected by LDH method.

[0134] Results: The DC vaccine can induce the...

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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 also discloses 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 a preparation method and use of tumor tissue whole antigens. Background technique [0002] At present, tumor vaccines are used to stimulate the generation of anti-tumor immunity in patients, that is, there are many methods for active immunotherapy of tumors, including tumor-specific / related antigenic proteins, tumor-specific / related antigenic polypeptides, plasmids expressing tumor antigens, Immune gene-modified tumor cells, dendritic cells (dendritic cells, DC) impacted by tumor antigens, etc. Among them, DCs challenged with tumor antigens are the most promising tumor vaccines, namely dendritic cell-based vaccines. [0003] DC vaccine is a new type of tumor treatment vaccine developed in the past ten years. The main mechanism is to immunize tumor patients with DCs carrying tumor antigens, and through mechanisms such as activating tumor-specific T cells in the body, to induce ...

Claims

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Application Information

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Patent Type & Authority Applications(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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