Manufacturing method of activated lymphocytes for immunotherapy

A technology of lymphocytes and cells, applied in the field of preparation of activated lymphocytes for immunotherapy, which can solve the problems of weak anti-cancer cell toxicity, difficulty in obtaining a large number of cells, and limitations in the clinical application of LAK cells

Inactive Publication Date: 2009-08-12
BINEX CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the early 1980s, Rosenberg's group completed LAK cells (lymphokine-activated killer cells that activate NK cells with IL-2) in melanoma, renal cell carcinoma, lymphoma, lung cancer, and colon cancer. cells) clinical trials [Rosenberg et al., 1985], but the clinical application of LAK cells is limited, because the anti-cancer cell toxicity of LAK cells is relatively weak, and it is difficult to obtain such cells in large quantities

Method used

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  • Manufacturing method of activated lymphocytes for immunotherapy
  • Manufacturing method of activated lymphocytes for immunotherapy
  • Manufacturing method of activated lymphocytes for immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Blood Collection and Separation of Lymphocytes

[0056] Collect 10-100ml of peripheral blood from human veins under sterile conditions. As for the blood collection container, a blood collection tube or bag containing an anticoagulant such as heparin or EDTA can be used. Then, the blood was injected into a 50ml centrifuge tube and mixed evenly with an equal amount of phosphate-buffered saline (PBS). Add Histopaque-1077 solution (Sigma) into a 50ml centrifuge tube, so that the ratio of Histopaque-1077 solution to blood diluted with PBS is 1:2-1:4. Then, slowly add blood diluted with PBS into the centrifuge tube so that the liquid level does not disperse. Then the mixture was centrifuged at room temperature at a speed of 400×g to separate the lymphocyte fraction. The section was then washed 3 times with an appropriate amount of phosphate-buffered saline. After final centrifugation and washing, the supernatant was removed, and the lymphocyte pellet was even...

Embodiment 2

[0057] Embodiment 2: the preparation of anti-CD3 antibody immobilization bottle

[0058] The antibody (Orthoclone OKT3 injection, manufactured by Ortho Biotechnology) was added to PBS to prepare a 10 ml anti-CD3 antibody solution with a concentration of 5 μg / ml, and the antibody solution was added to a culture bottle whose bottom surface area was 225 cm 2 , the solution can be evenly distributed on the bottom surface. On the next day, the antibody solution in the bottle was aspirated by a suction pump, and washed three times with PBS, thereby preparing an anti-CD3 antibody immobilized bottle.

Embodiment 3

[0059] Example 3: Culture of Activated Lymphocytes

[0060] In the present invention, the proliferation rate and activation of activated lymphocytes were compared under two different culture conditions. To this end, the lymphocyte suspension was added to 50 ml of each appropriate medium (G1: AIM-V (GIBGO, USA); G2: CeIIGro (CeIIGenix); G3: KBM (Kohjin Bio); G4: X-Vivo (Cambrex ))), each medium contains 1000 U / ml IFN-γ (Leucogen, LG Life Sciences) and 1-5% human serum. Then, each medium was incubated at 37 °C and 5% CO 2 Under conditions, cultured in cell culture flasks. After 24 hours of culture, the medium in each bottle was collected and transferred to a new 225-cm 2 500 U / ml IL-2 (Proleukin, CHIRON Company) and 50 ng / ml anti-CD-30 antibody (Orthoclone, Ortho Biotechnology Company) were added to each bottle. Meanwhile, the proliferation rate and activation of cells were compared between the case of culturing with the anti-CD3 antibody-immobilized flask prepared in Exam...

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PUM

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Abstract

Disclosed is a method for preparing activated lymphocytes, which comprises isolating lymphocytes from peripheral blood and proliferating and activating the isolated lymphocytes in vitro. According to the disclosed method, highly effective toxic cells can be prepared in large amounts by culturing human peripheral lymphocytes in the presence of an anti-CD3 antibody, IFN-gamma and IL-2. The activated lymphocytes proliferated according to the disclosed preparation method comprise both CD3-CD56+ (natural killer cell marker) cells that are the main components of LAK cells, and CD3+CD56+ cells that are the main components of CIK cells, and can be cultured in large amounts. Thus, the lymphocyte cells can show a significantly higher anticancer effect compared to when the LAK cells and the CIK cells are used alone.

Description

technical field [0001] The present invention relates to a method for preparing activated lymphocytes, more particularly to a method for preparing activated lymphocytes used as cellular immunotherapeutics. Lymphocytes, proliferate and activate the isolated lymphocytes in large quantities in vitro, and administer the activated lymphocytes to the human body from which the lymphocytes are derived; or by freezing the activated lymphocytes, and administering the frozen activated lymphocytes to lymphocytes Human body of cell origin. Background technique [0002] Human immune cells include natural killer (NK) cells and T cells, which recognize and eliminate transformed certain cells, such as cancer cells or cells infected with viruses. Therefore, utilizing the functions of those cells can prevent and treat these diseases. However, for cancer patients, it is difficult for immune cells to exhibit sufficient anti-cancer effects, because various anti-cancer therapies including surgery...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/02C12N5/0783
CPCA61K2035/124C12N2501/515C12N5/0646C12N2501/23C12N2501/24A61P31/12A61P35/00A61P37/04C12N5/0602C12N5/00
Inventor 朴淳元孙永玉孙哲勋朴秞秀潘贞花李炅奎张政洙姜治德金源硕安庆出李白天金柱寅朴恩卿崔成僖
Owner BINEX CO LTD
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