Process for production of cytokine-induced killer cells

A cytokine and cell killing technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of low proliferation of CIK cells and large physical burden, and achieve the effect of high therapeutic effect

Inactive Publication Date: 2012-07-11
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the proportion of CD3-positive CD56-positive cells contained in CIK cells prepared by traditional methods is generally about 20%. In addition, the proliferation of CIK cells is also very low, and the proliferation is about 10

Method used

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  • Process for production of cytokine-induced killer cells
  • Process for production of cytokine-induced killer cells
  • Process for production of cytokine-induced killer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 "CIK cell culture using anti-human CD3 antibody and Retronectin

[0082] (1) Separation and preservation of PBMC

[0083] From healthy people with informed consent, Donor A, Donor B, and Donor C, separate blood samples were taken (the so-called blood samples in this article are blood collection for the purpose of collecting monocytes). Dulbecco PBS (manufactured by Invitrogen or Nissui Pharmaceutical Co., Ltd.; hereinafter referred to as DPBS) or physiological saline containing 1% human serum albumin (preparation name: ブミネート; manufactured by Bectstar, hereinafter referred to as HSA) (Hereinafter referred to as 1% HSA / normal saline) Dilute the obtained components by about 2 times, and put them on Ficoll-Paque PREMIUM or Ficoll-Paque PLUS 15mL (both are produced by GE Healthcare Biosciences) The diluted components were collected and separated into 30 mL of blood, and centrifuged at 700×g for 20 minutes at room temperature. After centrifugation, the PBMC layer was r...

Embodiment 2

[0108] Example 2 "Culturing CIK cells using anti-human CD3 antibody and Retronectin (comparison of adding IFN-γ and using auto-irradiated cells)

[0109] (1) Preparation of autologous irradiated cells (hereinafter referred to as AIC)

[0110] After suspending the donor A-derived PBMC prepared in Example 1-(1) in 0.5% HAB / 0.2% HSA / GT-T503, an X-ray irradiation device was used to irradiate 3400R (29.8Gy) X-rays (hereinafter The cells after X-ray irradiation are called PBMC AIC). Press 1.06×10 of the prepared PBMC AIC 6 Cells / mL were resuspended in 0.5% HAB / 0.2% HSA / GT-T503.

[0111] (2) Cultivation of CIK cells

[0112] PBMC derived from Donor A prepared in Example 1-(1) was calculated as 1.06×10 6 Cells / mL were suspended in 0.5% HAB / 0.2% HSA / GT-T503 (referred to as the IFN-γ group without treatment the day before). However, a group was also established in which the same prepared PBMC was cultured overnight in the presence of IFN-γ (final concentration 1000 U / mL) on the day before the...

Embodiment 3

[0128] Example 3 "Culturing CIK cells with anti-human CD3 antibody and Retronectin (comparison of anti-human CD3 antibody concentration during restimulation)

[0129] (1) Preparation of PBMC AIC

[0130] Except for using donor B-derived PBMC or donor C-derived PBMC instead of donor A-derived PBMC, PBMC AIC was prepared in the same manner as in Example 2-(1).

[0131] (2) Cultivation of CIK cells

[0132] CIK cells were cultured in the same manner as in Example 2-(2). However, PBMC from Donor B or PBMC from Donor C (using the same donor-derived PBMC as the donor used in Example 3-(1)) was used for PBMC, and no IFN-γ treatment was performed the day before. In addition, when the anti-human CD3 antibody was re-stimulated on the 8th day of culture, the concentration of the anti-human CD3 antibody was 50 ng / mL or 200 ng / mL.

[0133] Continue the culture until the 14th day of the start of the culture. On the 14th day after the start of the culture, measure the number of viable cells by the t...

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Abstract

A process for producing cytokine-induced killer cells, comprising the steps of (a) culturing a cell mass that contains cells capable of being differentiated into CD3-positive CD56-positive cells and/or CD3-positive CD56-positive cells in the presence of a CD3 ligand, (b) culturing the cell mass produced in step (a) in the absence of a CD3 ligand, and (c) culturing the cell mass produced in step (b) in the presence of a CD3 ligand.

Description

Technical field [0001] The present invention relates to a method for preparing highly efficient cytokine-induced killer cells (CIK cells) in adoptive immunotherapy, CIK cells obtained by the preparation method, drugs containing the CIK cells, and the like. [0002] This application also claims the priority of Japanese Patent Application No. 2009-247347 filed on October 28, 2009, and the entire content of Japanese Patent Application No. 2009-247347 is incorporated into this application. Background technique [0003] In recent years, treatment methods that have a heavy physical burden on patients such as drug therapy and radiotherapy have been re-evaluated, and interest in immunotherapy with a lighter burden has increased. The therapy includes, for example, adoptive immunotherapy in which the extracted immune-related cells are cultured in vitro to increase the number of cells, and / or strengthen the activity related to the therapeutic effect and transplant it into the patient. [0004]...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/14A61K35/28A61P1/16A61P31/04A61P31/06A61P31/10A61P31/16A61P31/18A61P37/04C12N5/00A61K35/17C12N15/09
CPCC12N2501/24C12N2501/515C12N2501/23C12N5/0636A61K35/12A61P1/16A61P31/04A61P31/06A61P31/10A61P31/16A61P31/18A61P37/04
Inventor 神芽衣子出野美津子榎龙嗣
Owner TAKARA HOLDINGS
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