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Method of proliferating LAK cell

A cell and proliferation factor technology, applied in the field of LAK cell proliferation, can solve the problems of lymphocyte therapy not being effective, not showing antigen specificity, MHC binding, and expensive

Inactive Publication Date: 2009-07-29
ARIGEN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, there is a problem with the anti-CD3 antibody that the CD3 protein as an antigen differs depending on the source species such as dogs, cats, domestic animals, humans, etc., so it is necessary to prepare anti-CD3 antibodies for each species to be treated
In addition, the anti-CD3 antibody, especially the anti-CD3 antibody for human commonly used at present, is expensive
Therefore, LAK therapy and CTL therapy, etc. have the problem of activating autologous lymphocytes. Leaving aside human beings, it is difficult to apply them to non-human animals from an economical point of view.
[0008] In addition, even if only γδ T cells are proliferated, γδ T cells do not show antigen specificity and MHC restriction as described above, so there is a problem that autologous lymphocyte activation therapies such as LAK therapy and CTL therapy are not very effective

Method used

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  • Method of proliferating LAK cell
  • Method of proliferating LAK cell
  • Method of proliferating LAK cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0087] Using concanavalin A as a lectin and human interleukin-2 as a growth factor having interleukin-2-like activity, the optimal growth conditions of LAK cells were investigated.

[0088] 1. Optimization of Concanavalin A Concentration

[0089] (1) The collection of initial samples

[0090] 30 ml of whole blood (prevented with heparin from clotting) was drawn from the jugular vein of a doggy (6 years old) with a syringe. After centrifugation, add 10 times the amount of (a) NH 4 Cl buffer (NH 4 Cl: 0.83g / 100ml distilled water), (b) Tris base (20.6g / 1000ml distilled water, pH7.2), (c) (a) and (b) described in (c) mixed in a 9:1 filter-sterilized solution , placed in ice for 5 minutes (stirring frequently) to rupture the red blood cells contained in the blood.

[0091] 3 ml of specific gravity solution (for canine lymphocyte separation, lymphocyte (Lympholyte) manufactured by Cedarlane Co., Ltd.) was evenly put into 2 centrifuge tubes, and the ruptured blood was slowly layere...

Embodiment 2

[0128] The effect of different sera used on the proliferation of LAK cells was investigated by changing the sera used to autoserum, fetal bovine serum, and cat serum. Specifically, follow the procedures below to investigate.

[0129] 1. When using your own serum

[0130] (1) Experimental method

[0131] By the same method as in Example 1, 1 ml of 4 systems were obtained from 4 dogs (Miniature Pinscher, 2 years old; Hybrid, 1 year old; Pomeranian, 10 years old; Hybrid, 3 years old) Cell suspension (cell density: 1.5×10 6 cells / ml). Add culture medium to the obtained cell suspensions of each system to adjust the number of cells to 1×10 5 cells / ml, add 3ml (3×10 5cells / well), Concanavalin A at a final concentration of 5 μg / ml, autoserum at a final concentration of 5%, and interleukin-2 at a final concentration of 750 U / ml were added respectively. In addition, in the same manner as in Example 1, the number of cells at the start day of cell proliferation was measured using a ...

Embodiment 3

[0157] Changes in cell distribution due to proliferation were investigated by flow cytometry. Specifically, by the proliferation method of the present invention, for CD8 cells related to cellular immunity as αβ T cells + Cellular and CD4 associated with humoral immunity + The number of cells and the change in the ratio of the cells were investigated as follows.

[0158] (1) Experimental method

[0159] By the same method as in Example 1, 5 ml of cell suspension (cell density: 2~4×10) of 3 systems (for example, case) were obtained from 3 dogs (6 years old). 7 cells / ml). Add culture medium to the obtained cell suspensions of each system to adjust the number of cells to 1×10 6 Cells / ml, add 2ml per well (2×10 6 cells / well), add concanavalin A with a final concentration of 5 μg / ml, canine serum with a final concentration of 5% (taken from a dog for blood transfusion), fetal bovine serum with a final concentration of 10%, and concanavalin A with a final concentration of 750U / m...

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Abstract

The present application aims at providing a treatment method effective for various cancers, immune deficiency diseases and infections, and a method of proliferating / activating cells associated therewith, which can be performed at such low costs that the methods can be applicable to nonhuman animals. A method of proliferating / activating cells of the present application comprises steps of, during the cell culture, supplementing a plant lectin (such as concanavalin A) and a growth factor having interleukin-2-like activity to the culture medium. Accordingly, the present method can proliferate / activate, on a preferential basis, a-type T cells associated with cell-mediated immunity for cancers, immunodeficiency diseases and infectious diseases.

Description

technical field [0001] The present invention relates to a method for proliferating LAK cells, a cell preparation including the proliferated cells, a method for treating non-human animal cancers, immune diseases, and infectious diseases using the cell preparation, and a culture kit for the proliferation of LAK cells. Background technique [0002] Conventional methods for treating cancer and tumors include surgery in which a lesion is excised, chemotherapy in which an anticancer agent is administered, and radiation therapy in which radiation is irradiated to a lesion. [0003] However, these treatment methods have disadvantages such as heavy burden on the body of the patient and side effects caused by chemicals and radiation. Therefore, since around 1980, LAK (lymphokine-activated killer, lymphokine-activated killer cell) therapy and CTL (cytotoxic T lymphocyte, cytotoxic T lymphocyte) therapy, which are the fourth treatment methods, have been used to activate autolymphocyte t...

Claims

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Application Information

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IPC IPC(8): C12N5/06A61K35/14A61P31/00A61P35/00A61P37/02C12N5/0783
Inventor 渡来仁西川茂
Owner ARIGEN PHARMA INC
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