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Tumor specific target and application thereof in preparing preparation for cellular immunotherapy

A tumor-specific, cellular immune technology, applied in animal cells, anti-tumor drugs, vertebrate cells, etc., can solve problems such as staying

Active Publication Date: 2015-06-10
河北博海生物工程开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the clinical cellular immunotherapy technology basically stays at the level of LAK, NK, CIK, and DC, while the adenovirus-transfected antigen-loaded and CAR-T technologies contain gene technology, which is controversial in terms of ethics.

Method used

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  • Tumor specific target and application thereof in preparing preparation for cellular immunotherapy
  • Tumor specific target and application thereof in preparing preparation for cellular immunotherapy
  • Tumor specific target and application thereof in preparing preparation for cellular immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: In vitro amplification and activation method for tumor-specific dendritic cell (DC) targets

[0019] 1. Specimen collection:

[0020] The blood picture of melanoma patients is required to be within the normal range (WBC: 4-10×10 9 , LYM%: 20%-40%), the fluctuation does not exceed 5%, the circulation volume of peripheral blood by machine sampling is 1000-4000ml, the number of mononuclear cells: more than 1×10 9 . Blood drawing method 50-100ml, the number of mononuclear cells: more than 1×10 6 .

[0021] The preferred anticoagulant is heparin, followed by sodium citrate, and EDTA is prohibited.

[0022] The collected specimens should not be stored for more than 6 hours, and cell preparation should be performed as soon as possible. If the specimen is stored for more than 2 hours before preparation, the specimen should be stored at 4°C.

[0023] 2. Preparation before operation

[0024] Wipe the collection bag containing the suspension of PBMC peripheral bl...

Embodiment 2

[0055] Example 2: Test method for detection of cell positive rate by target monoclonal antibody

[0056] Positive detection steps of indirect immunofluorescence on the surface of peripheral blood DC cells in patients with colon cancer

[0057] 1. Take the peripheral blood DC cells (1×10 7 / ml) 200 μl, divided into two tubes, one tube for each experimental control (100 μl / tube).

[0058] 2. Centrifuge and wash once with PBA,

[0059] 3. Add 20ul of the target monoclonal antibody diluted with PBA to the experimental tube, add 20ul of PBA to the control tube, gently pipette and mix, and incubate at 4°C or on ice for 1.5-2h. Centrifuge and discard the supernatant.

[0060] 4. Centrifuge and wash once with 1ml of PBA to remove excess unbound specific antibody.

[0061] 5. 20ul of fluorescein-labeled secondary antibody appropriately diluted in PBA. Mix by pipetting and incubate at 4°C for 30 min, protected from light. (PBA: PBS plus 1-2% bovine serum albumin, plus 0,1% sodium ...

Embodiment 3

[0066] Embodiment 3: the test method of DC cell marker antibody detection cell positive rate

[0067] Positive detection steps of indirect immunofluorescence on the surface of peripheral blood DC cells in patients with melanoma

[0068] 1. Take the peripheral blood DC cells (1×10 7 / ml) 200 μl, divided into two tubes, one tube for each experimental control (100 μl / tube).

[0069] 2. Centrifuge and wash once with PBA;

[0070] 3. Add 20 μl each of FITC-CD86, PE-HLA-DR, and APC-CD11c to the experimental tube, and add 20 μl each of FITC-mouse IgG1, PE-mouse IgG1, and APC-mouse IgG1 to the control tube.

[0071] 4. Gently blow and beat to mix well. After each tube is fully mixed, incubate in the dark for 30 minutes.

[0072] 5. After washing with PBS, test on the machine.

[0073] 6. See the test results image 3 ,analyse as below:

[0074] Experimental tube:

[0075] HLA-DR + Cells: 99.74%

[0076] CD11c + Cells: 98.83%

[0077] CD86 + Cells: 99.69%

[0078] HLA-DR ...

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Abstract

The invention relates to the technical field of biology, and discloses a tumor specific target and application thereof. The target amino acid sequence is SAKYGVRKF. The tumor specific target can implement extraneous effective amplification and activation of dendritic cells. The provided T lymphocyte has specific killing effects on tumors, and is a cellular immunotherapy technique which most conforms to the cellular immunology principle at present.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to tumor-specific targets and applications thereof. Background technique [0002] Tumor is a kind of disease that seriously endangers human health. The environmental pollution caused by human's transformation of nature has greatly increased the number of malignant tumor patients worldwide. However, the traditional treatment methods such as surgery, chemotherapy, and radiotherapy, due to the lack of targeting, also greatly damage the normal tissue cells of the body while treating tumors, and cannot achieve good anti-tumor effects. [0003] Cellular immunotherapy is an emerging treatment mode with significant curative effect, and it is a new treatment method based on autoimmunity. It uses biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then return to the body. The method of transfusion into the patient's body to stimulate / enhanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N5/0784C12N5/0783A61K39/00A61P35/00
Inventor 李彬
Owner 河北博海生物工程开发有限公司
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