Method of extensive culture of antigen-specific cytotoxic T cells

a cytotoxic t cell and culture method technology, applied in the field of inducing, maintaining and expanding cytotoxic t cells, can solve the problems of inability to obtain the necessary cell count, inability to proliferate t cells to 10sup>9 /sup>to 10 and eventually decrease the cell coun

Inactive Publication Date: 2008-07-10
SAGAWA HIROAKI +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0254]According to the present invention, there is provided a method for inducing, maintaining and expanding CTL having antigen-specific cytotoxic activity at a high level. This method is extremely useful in the field of cell remedy such as adoptive immunotherapy requiring a large amount of CTLs. In addition, since the CTL prepared by this method is prepared by a safe method, the CTL can be a cell medicament having very high safety.

Problems solved by technology

In other words, in adoptive immunotherapy, it can be said that it is a major problem to obtain the above cell count in vitro in a short period of time.
However, in this method, the cell count may temporarily be increased, but the cell count is eventually decreased, and necessary cell count cannot be obtained.
However, it is impossible to proliferate T cell to 109 to 1010 cells by this method.
This cell count is enormous, considering that it usually takes about 24 hours for a single cell to be divided and proliferated into two cells.
However, the former is a method for obtaining T cell which is non-specific for an antigen, and in the latter, antigen specificity is very low, if any, because activated polyclonal lymphocyte population is used.
Therefore, the effectiveness of LAK cell or TIL obtained by the above-mentioned methods is problematic.
Therefore, LAK cell, TIL and the method for culturing T cell at a low density are problematic in both aspects of actual use and usefulness.
However, there is a serious problem in these methods.
Specifically, it takes about 3 months to obtain 1×109 cells / ml of antigen-specific CTLs, during which time the symptoms of the patient advance, so that it is difficult to appropriately treat the disease depending on the situation.
However, there is a problem as described below.
However, there are problems that risk of admixing EBV-transformed B cell (EBV-B cell) into T cell is not deniable (problem in safety); that a large amount of PBMC (PBMC in an amount of about 40 times the number of antigen-specific CTL required) is required as feeder cell; that the antigen-specific cytotoxic activity of the expanded CTL cannot be sufficiently satisfactory; that the antigen-specific cytotoxic activity possessed by T cell is decreased with the cell proliferation when CTL is allowed to proliferate using a T cell population other than the T cell clone; and the like.
In other words, in a conventional method for preparing antigen-specific CTL, there have not been solved the problems essential to adoptive immunotherapy in which CTL having an antigen-specific cytotoxic activity effectively used in the treatment, is prepared in a sufficient amount for a short period of time.

Method used

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  • Method of extensive culture of antigen-specific cytotoxic T cells
  • Method of extensive culture of antigen-specific cytotoxic T cells
  • Method of extensive culture of antigen-specific cytotoxic T cells

Examples

Experimental program
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preparation example 1

[0094]Kjellmaniella crassifolia was sufficiently dried, and thereafter 20 kg of the dried product was powdered with a free mill (manufactured by Nara Kikai Seisakusho). In 900 liters of tap water was dissolved 7.3 kg of calcium chloride dihydrate (manufactured by Nippon Soda Co., Ltd.), and 20 kg of the powdered product of Kjellmaniella crassifolia was then mixed therewith. The resulting mixture was heated for 40 minutes by blowing steam until the liquid temperature was raised from 12° to 90° C. Thereafter, the mixture was kept at 90° to 95° C. for 1 hour under stirring, and then cooled, to give 1100 liters of a cooled product. Subsequently, the cooled product was subjected to solid-liquid separation with a solid-liquid separator (manufactured by West Farrier Separator, Model: CNA), to give about 900 liters of supernatant after solid-liquid separation. The amount 360 liters of the supernatant after solid-liquid separation was concentrated up to a volume of 20 liters with FE10-FC-FUS...

preparation example 2

[0095]Seven grams of the dried product of the fucoidan described in Preparation Example 1 was dissolved in 700 ml of a 20 mM imidazole buffer (pH 8) containing 50 mM sodium chloride and 10% ethanol, and insoluble substances were removed by centrifugation. The supernatant after centrifugation was applied onto a DEAE-Cellulofine A-800 column (φ11.4 cm×48 cm) (manufactured by Seikagaku Corporation) equilibrated with the same buffer, and then washed with the same buffer. The elution was carried out with a concentration gradient of from 50 mM to 1.95 M sodium chloride (250 ml per fraction). A total sugar content and an uronic acid content were determined by the phenol-sulfuric acid method and the carbazole-sulfuric acid method, to give Fractions 43 to 49, Fractions 50 to 55, and Fractions 56 to 67, in the order of elution. Next, these fractions were desalted by electrodialysis, and thereafter lyophilized, to give each of Fraction I (340 mg) from Fractions 43 to 49, Fraction II (870 mg) f...

preparation example 3

[0096](1) A 2-liter Erlenmeyer flask was charged with 600 ml of a culture medium comprising an artificial sea water (manufactured by Jamarin Laboratory), pH 8.2, containing 0.25% glucose, 1.0% peptone, and 0.05% yeast extract, and then sterilized (at 120° C. for 20 minutes). Alteromonas sp. SN-1009 (FERM BP-5747) was inoculated into the culture medium, and cultured at 25° C. for 26 hours, to give a seed culture medium. A 30-liter jar fermentor was charged with 20 liters of a culture medium comprising an artificial sea water, pH 8.0, containing 1.0% peptone, 0.02% yeast extract, 0.2% sulfated polysaccharide described in item (2) of Example 2 described below, and 0.01% defoaming agent (manufactured by Shin-Etsu Chemical Co., Ltd., KM70), and sterilized at 120° C. for 20 minutes. After cooling, a 30 L jar fermentor was charged with 600 ml of the above-mentioned seed culture medium, and cultured at 24° C. for 24 hours under the conditions of 10 liters of aeration per minute and a stirri...

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Abstract

The present invention provides methods for inducing, maintaining and expanding CTL (cytotoxic T cell) having an antigen-specific cytotoxic activity at a high level, which is useful in the adoptive immunotherapy, by using as an effective ingredient at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides, and salts thereof. The above-mentioned compounds include fucoidans, heparins, alginic acid, chondroitin sulfate A, chondroitin sulfate B, pectic acid, hyaluronic acid, degradation products of fucoidans, sulfated glucose, sulfated fucose and salts thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a Divisional of co-pending application Ser. No. 10 / 344,534, filed on Feb. 12, 2003, which is a National Phase under 35 U.S.C. § 371 of PCT International Application No. PCT / JP01 / 07032 which has an International filing date of Aug. 15, 2001, which designated the United States of America, which claims priority to Japanese Patent Application No. 2000-247072 filed Aug. 16, 2000, the entire contents of which are hereby incorporated by reference.TECHNICAL FIELD[0002]The present invention relates to methods for inducing, maintaining and expanding cytotoxic T cell having an antigen-specific cytotoxic activity, which is useful in the medical field.BACKGROUND ART[0003]A living body is protected from foreign substances mainly by an immune response, and an immune system has been established by various cells and the soluble factors produced thereby. Among them, leukocytes, especially lymphocytes, play a key role. The lymphocytes are...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N63/00C12Q1/20C12N5/00A61K35/14A61K35/16A61K35/17A61K39/00A61K39/145A61P37/04C12N5/07C12N5/0783
CPCA61K39/0011A61K39/145A61K2039/5158C12N2760/16134C12N5/0636C12N2501/515C12N2501/90A61K2039/55583A61K39/12A61P31/12A61P35/00A61P37/04C12N5/00
Inventor SAGAWA, HIROAKIIDENO, MITSUKOKATO, IKUNOSHIN
Owner SAGAWA HIROAKI
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