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Method for activating LAK (lymphokine activated killer)

A technology for killing cells and lymphokines, applied in the direction of cell culture active agents, animal cells, vertebrate cells, etc., which can solve the problems of weak lethality, large infusion, high cost, etc.

Inactive Publication Date: 2018-11-06
淮安诺康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, LAK is not lethal, and clinical application requires a large amount of infusion
Moreover, its expansion ability is limited, and large doses of IL-2 need to be applied at the same time as cell infusion, resulting in obvious toxic side effects. The most common and serious side effect is capillary leak syndrome, which is mainly manifested as generalized edema and multiorgan dysfunction, which can cause pleural and peritoneal effusions, pulmonary interstitial edema, and congestive heart failure
[0004] At present, LAK cells need to be cultured in vitro for at least 3 to 7 days. The process is long, expensive, and easily polluted, which limits clinical application. Therefore, the research on simple and fast induction culture has never stopped.

Method used

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  • Method for activating LAK (lymphokine activated killer)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the preparation of polypeptide

[0051] 1. Defat wheat germ

[0052] Use defatted wheat germ, or directly purchase defatted wheat germ for subsequent experiments. Degreasing conditions: extractant: petroleum ether (60°C-90°C), water bath temperature: 65°C, solid-liquid ratio: 1:8 (g / mL), degreasing time: 4h.

[0053] The defatted wheat germ is pulverized into 80 meshes to obtain defatted wheat germ powder, which is set aside.

[0054] 2. Hydrolysis and purification of peptides

[0055] The defatted wheat germ powder was treated with papain (enzyme activity of 800,000 U / g) + neutral protease (enzyme activity of 200,000 U / g) at a compound ratio of 3:1 (mass ratio), pH of 6.0, and temperature Enzymolysis under the conditions of 55°C, substrate mass concentration 40g / L, enzyme addition amount 5% (100g substrate plus 5g enzyme), enzymolysis time 8h, after enzymolysis, the temperature was raised to 90°C and kept for 20min for enzymatic inactivation treatment ...

Embodiment 2

[0059] Embodiment 2: LAK cell rapid activation method

[0060] 1. Experimental materials

[0061] RPMI 1640 medium and 10% fetal bovine serum were purchased from Gibco.

[0062] rIL-2 was purchased from Beijing Sihuan Biopharmaceutical Company.

[0063] 2. Experimental method

[0064] 1. Umbilical cord blood collection and mononuclear cell isolation

[0065] The closed collection method was used to collect cord blood from 100 cases of healthy neonates born in full-term normal delivery, and the parents of the prenatal examinations had no family history of genetic diseases. It was collected from the side away from the neonatal side of the umbilical cord stump before delivery. After collection, they were stored in a refrigerator at 4°C, and separated and frozen within 24 hours.

[0066] With reference to the technical handbook in this field (Shen Caring etc., Modern Immunology Experimental Technology, Hubei Science and Technology Press, 1998, 172-175 pages), adopt Ficoll dia...

Embodiment 3

[0080] Embodiment 3: LAK cell rapid activation method

[0081] 1. Experimental materials

[0082] RPMI 1640 medium and 10% fetal bovine serum were purchased from Gibco.

[0083] rIL-2 was purchased from Beijing Sihuan Biopharmaceutical Company.

[0084] 2. Experimental method

[0085] 1. Umbilical cord blood collection and mononuclear cell isolation

[0086] The closed collection method was used to collect cord blood from 100 cases of healthy neonates born in full-term normal delivery, and the parents of the prenatal examinations had no family history of genetic diseases. It was collected from the side away from the neonatal side of the umbilical cord stump before delivery. After collection, they were stored in a refrigerator at 4°C, and separated and frozen within 24 hours.

[0087] With reference to the technical handbook in this field (Shen Caring etc., Modern Immunology Experimental Technology, Hubei Science and Technology Press, 1998, 172-175 pages), adopt Ficoll dia...

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Abstract

The invention discloses a method for activating LAK (lymphokine activated killer). The method includes: modulating mononuclear cells with an RPMI 1640 medium (10% FCS-RPMI 1640) containing 10% of fetal calf serum to 2X106 / mL; inoculating the medium in culture containers of different volumes according to the total number of cells and meanwhile, adding enzymolysis polypeptide A, enzymolysis polypeptide C or enzymolysis polypeptide D with the final concentration of 40-60 microns / mL and rIL-2 of 500IU / mL and placing under the conditions of 37DEG C and 5% of CO2 for cultivation of 24 hours. The rapid activating method has the advantages that on the premise that rIL-2 is not used extensively, the enzymolysis polypeptide A, enzymolysis polypeptide C or enzymolysis polypeptide D are added in the medium, and LAK is activated rapidly after only 24 hours.

Description

technical field [0001] The invention belongs to the field of biology and relates to a method for activating LAK of lymphokine-activated killer cells. Background technique [0002] Immunotherapy is an emerging tumor treatment method, which has been listed as the fourth treatment method after surgery, radiotherapy, and chemotherapy, and is gradually playing an important role in the comprehensive treatment of tumors. In 2010, the US FDA approved Provenge, the world's first cellular immunotherapy product, to go on the market, fully demonstrating the value of cellular immunotherapy in tumor treatment. [0003] Lymphokine-activated killer cells (LAK) were developed in the 1980s by Rosenberg et al. Its essence is NK and T cells with tumoricidal activity activated by IL-2. In November 1984, Rosenberg's research group was approved by the US FDA to use LAK cells for clinical treatment for the first time. The therapy has been shown to be effective in patients with metastatic renal ce...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12P21/06C07K1/34
CPCC12N5/0638C07K1/34C12N2501/2302C12N2501/999C12P21/06
Inventor 何英广马思航
Owner 淮安诺康生物科技有限公司
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