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 and heavy physical burden of CIK cells, and achieve high therapeutic effect

Inactive Publication Date: 2013-09-11
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 times after 3 weeks of culture.
Therefore, in order to culture a large number of CIK cells, blood components are often collected from patients, and there is a problem of heavy physical burden (for example, non-patent documents 2 and 3)

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) Isolation and preservation of PBMC

[0083] Component blood collection was performed from healthy human donors A, B and C who had obtained informed consent (the so-called component blood collection in this paper refers to blood collection for the purpose of collecting mononuclear cells). Dulbecco PBS (manufactured by Invitrogen Corporation or Nissui Pharmaceutical Co., Ltd.; hereinafter referred to as DPBS) or physiological saline containing 1% human serum albumin (preparation name: ブミンネート; manufactured by Bakster Corporation, hereinafter referred to as HSA) (Hereafter referred to as 1% HSA / Normal Saline) The obtained components were blood-diluted approximately 2-fold, and placed on Ficoll-Paque PREMIUM or Ficoll-Paque PLUS 15mL (either of which is produced by GE Healthcare Biosciences) The diluted components were collected into separate layers of 30 mL each, and centrifuged at 700×g ...

Embodiment 2

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

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

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

[0111] (2) Culture of CIK cells

[0112] The PBMC derived from donor A prepared in Example 1-(1) was divided into 1.06×10 6 Cells / mL were suspended in 0.5% HAB / 0.2% HSA / GT-T503 (referred to as IFN-γ untreated group the day before). However, a group was also established in which PBMCs prepared in the same way were cultured overnight in the presence of IFN-γ (final concentration 1000 U / mL) the day b...

Embodiment 3

[0128] Example 3 Culturing CIK cells using anti-human CD3 antibody and Retronectin (comparison of anti-human CD3 antibody concentration during re-stimulation)

[0129] (1) Preparation of PBMC AIC

[0130] PBMC AIC was prepared in the same manner as in Example 2-(1), except that donor B-derived PBMCs or donor C-derived PBMCs were used instead of donor A-derived PBMCs.

[0131] (2) Culture of CIK cells

[0132] CIK cells were cultured in the same manner as in Example 2-(2). However, PBMCs derived from donor B or PBMCs derived from donor C (the same donor-derived PBMCs as used in Example 3-(1) were used) were used as PBMCs, 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 from the start of the culture, the concentration of the anti-human CD3 antibody was 50 ng / mL or 200 ng / mL.

[0133] Continue to culture until the 14th day after the start of culture. On the 14th day after the start of culture, ...

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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; and others.

Description

technical field [0001] The present invention relates to a preparation method of highly efficient cytokine-induced killer cells (CIK cells) in adoptive immunotherapy, CIK cells obtained by the preparation method, medicines 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 contents of Japanese Patent Application No. 2009-247347 are incorporated in this application. Background technique [0003] In recent years, treatment methods such as drug therapy and radiation therapy that have a heavy physical burden on patients have been re-evaluated, and attention to immunotherapy with a lighter burden has increased. The therapy includes, for example, adoptive immunotherapy in which the removed immune-related cells are cultured in vitro to increase the number of cells, and / or enhance the activity related to the therapeutic effect and transplanted into the pati...

Claims

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Application Information

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