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Method for amplifying and activating lymphocyte by using CD8 alpha-interleukin 21-CD137 compound

A 21-CD137, CD137 technology, applied in the field of immunology, can solve the problems of large economic burden for patients, limited LAK cell ploidy, and difficulty in obtaining a large number of LAK cells

Active Publication Date: 2011-08-17
杭州中赢生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The number of LAK cells is directly related to the curative effect. The problem in the treatment is that it is very difficult to obtain a large number of LAK cells
High doses of interleukin 2 are expensive and a great financial burden on patients
The current study demonstrates that the limited fold-fold growth of LAK cells activated by IL-2 is associated with decreased telomerase activity in cells following IL-2 expansion

Method used

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  • Method for amplifying and activating lymphocyte by using CD8 alpha-interleukin 21-CD137 compound
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  • Method for amplifying and activating lymphocyte by using CD8 alpha-interleukin 21-CD137 compound

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Experimental program
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Effect test

Embodiment 1

[0043] Transmembrane interleukin 21-CD137 complex expands LAK cells in vitro.

[0044] NK cells from healthy donors were cultured in RPMI1640 supplemented with 10% human serum. K562 cells expressing transmembrane interleukin-21-CD137 complex (irradiated, 100Gy) and low-dose interleukin-2 were added to the culture medium and cultured for 7 days. Centrifuge after 7 days, resuspend with an equal amount of culture medium, add irradiated K562 cells, and culture for another 7 days ( figure 1 ). High-dose interleukin-2 (200 units / ml) served as the control group. Such as figure 2 As shown in A, the number of LAK cells was significantly increased under the joint action of transmembrane interleukin-21-CD137 complex and low-dose interleukin-2. An increase in cell number can be measured by the insertion of thymidine. Cells were cultured for 12 hours after thymidine insertion before measurements of changes in cell number were started. Under the action of the transmembrane interleuki...

Embodiment 2

[0046] Transmembrane interleukin 21-CD137 complex expands unpurified peripheral blood lymphocytes

[0047] The transmembrane interleukin 21-CD137 complex can expand not only purified NK cells, but also unpurified lymphocytes. PBM from healthy donors were cultured in RPMI1640 supplemented with 10% human serum. K562 cells expressing transmembrane interleukin-21-CD137 complex (irradiated, 100Gy) and low-dose interleukin-2 were added to the culture medium and co-cultured for 7 days. Centrifuged after 7 days, resuspended with an equal amount of culture medium, added K562 cells irradiated by 100Gy, and cultured for another 7 days ( figure 1 ). High-dose interleukin-2 (200 units / ml) served as the control group. Such as figure 2 As shown in B, the number of LAK cells was significantly increased under the joint action of transmembrane interleukin-21-CD137 complex and low-dose interleukin-2. The number of LAK cells entered the logarithmic growth phase at 7 days, and increased abou...

Embodiment 3

[0050] Detection of Telomerase Activity and STAT3 Phosphorylation in LAK Cells

[0051] The amplified cells were disrupted by repeated freezing and thawing with liquid nitrogen, centrifuged at 10,000 g, and the supernatant was taken, and the telomerase activity of the amplified LAK cells was detected by the TRAP method. 50 μl TRAP system contains 20 mmol / L Tris-HCL (pH8.3), 1.5 mmol / L MgCL, 63 mmol / L, KCL, 0.005% Tween-20, 1 mmol / L EDTA, 50 Millimolar / L dNTP, 0.1 μg tS, 1 μg T4, 0.1 mg / ml bovine serum albumin, 1-2 μl CHAPS, cell extract (including 6 μg) protein, 0.2-0.4 μl [α- 32 P]dGTP. Reaction conditions: 10 minutes at 23°C, 3 seconds at 94°C, add Taq enzyme, 30 seconds at 94°C, 30 seconds at 50°C, 1.5 minutes at 72°C, 27 cycles of amplification, take 25 μl of product for 15% PAGE gel electrophoresis. Autoradiography on X-ray film after PAGE gel electrophoresis for 8 hours to more than two days. Compared with before amplification, the high dose of interleukin 2 in the c...

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Abstract

The invention relates to the field of immunology, in particular to a method for amplifying and activating a natural killer (NK) cell into a lymphokine-activated killer (LAK) cell by using a CD8 alpha-interleukin 21-CD137 compound. The method disclosed by the invention comprises the following steps of: forming the CD8 alpha-interleukin 21-CD137 compound by using CD8 alpha, interleukin 21 and a CD137 functional polypeptide, making an exogenous expression vector enter a host K562 cell, then activating a promoter and culturing a cell to obtain a cell for expressing a transmembrane interleukin 21-CD137 compound; and purifying the compound in the conventional way, and amplifying and activating a lymphocyte by using the purified compound to generate the LAK cell. The method has the advantage that: the LAK cell cultured and amplified by using the transmembrane interleukin 21-CD137 compound and a small dose of interleukin 2 is used for enhancing the immunity of a patient to help the patient resist tumors, viruses and bacteria. The method has a wide clinical using prospect.

Description

technical field [0001] The invention relates to the field of immunology, specifically refers to the use of CD8α-interleukin 21-CD137 complex transfected K562 cells and low concentration of interleukin 2 to work together to expand and activate natural killer cells (NK) to become lymphokine-activated killer cells ( LAK) cells. technical background [0002] In 1982, Grimm et al. first reported that adding interleukin 2 to peripheral blood lymphocytes and culturing them in vitro for 4-6 days could induce a kind of non-specific killer cells, which could kill a variety of tumor cells insensitive to NK. Such cells are called lymphokine-activated killer cells (LAK). In November 1984, under the approval of the US Food and Drug Administration (FDA), the Rosenberg research group first applied IL-2 and LAK to treat 25 patients with renal cell carcinoma, melanoma, lung cancer, colon cancer and other tumors. Eleven of them had tumors that shrank by more than 50%, and one melanoma comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0781
Inventor 吴忠福徐以兵董生聚
Owner 杭州中赢生物医疗科技有限公司
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