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WO2006006720A1 - MÉTHODE POUR FAIRE LA CULTURE DES CELLULES ϜδT, LES CELLULES ϜδT ET UN REMÈDE/TRAITEMENT PRÉVENTIF - Google Patents

MÉTHODE POUR FAIRE LA CULTURE DES CELLULES ϜδT, LES CELLULES ϜδT ET UN REMÈDE/TRAITEMENT PRÉVENTIF Download PDF

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Publication number
WO2006006720A1
WO2006006720A1 PCT/JP2005/013218 JP2005013218W WO2006006720A1 WO 2006006720 A1 WO2006006720 A1 WO 2006006720A1 JP 2005013218 W JP2005013218 W JP 2005013218W WO 2006006720 A1 WO2006006720 A1 WO 2006006720A1
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Prior art keywords
cells
acid
concentration
culturing
peripheral blood
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PCT/JP2005/013218
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English (en)
Japanese (ja)
Inventor
Mie Nieda
Masashi Takahara
Masato Mutou
Nami Satou
Kazuhiro Kakimi
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Medinet., Co.Ltd
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Priority to JP2006529212A priority Critical patent/JPWO2006006720A1/ja
Publication of WO2006006720A1 publication Critical patent/WO2006006720A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells

Definitions

  • the present invention relates to a method for culturing ⁇ 5 ⁇ cells. Further, the present invention relates to a cultivated cell (5 ⁇ cells, a therapeutic-preventive agent containing a cell cultured by the culture method.
  • cancer malignant neoplasm
  • T cells there is a therapy that activates T cells called L ⁇ (1 ymp h o kine e-a c t i v a t e d k i l l e r; lymphokine-activated killer) therapy.
  • L ⁇ (1 ymp h o kine e-a c t i v a t e d k i l l e r; lymphokine-activated killer
  • T cells There are two types of T cells: ⁇ / 3-type TCR (T cellreceptor)] 3 T cells and ⁇ -type TCR-expressing ⁇ cells. This is a therapy that activates ⁇ cells.
  • ⁇ 3 ⁇ cells are those that are mainly responsible for acquired immunity.
  • ⁇ cells are cells responsible for innate immunity. Recently, it has been found that it has cytotoxic activity (non-specific activity) against cancer cells, and research on immunotherapy using the strong antitumor activity of ⁇ cells has been conducted.
  • ⁇ ⁇ ⁇ cells recognize and activate non-peptidic antigens, and as a non-peptidic antigen, for example, alkylamines can be stimulated with bisphosphonates to activate and / or proliferate.
  • a non-peptidic antigen for example, alkylamines can be stimulated with bisphosphonates to activate and / or proliferate.
  • ⁇ cells are usually present in only 1 to 5% of peripheral blood, even if a small amount of blood is collected to activate and / or proliferate ⁇ cells, the purity and number of cells sufficient for treatment There is a problem that cannot be secured. Also treatment If the amount of blood collected from a patient is increased in order to ensure sufficient purity and cell count, there is also a problem that a great burden is placed on the patient.
  • CD 56-positive cells 5T cells
  • CD 56-negative cells ⁇ T cells positive ⁇ T cells are known to have higher cytotoxic activity than CD 56 negative ⁇ ⁇ cells (Fu j imiya, Y. eta 1. Clinical Cancer Re search Vo l. 3, 6 33— 643, Ap ril 1997), and is expected to be applied to the treatment of cancer and infectious diseases.
  • JP-A-2001-314183 describes therapeutic agents and examples using a cell group containing 50% or more of CD56-positive cells.
  • the CD56-positive cells used here are A ⁇ cells and natural killer cells (hereinafter referred to as ⁇ cells) are mixed, and only about 10% of ⁇ ⁇ 5 ⁇ cells are included. Disclosure of the invention
  • the present invention has been made in view of the above circumstances, and is capable of selectively activating and cultivating or proliferating cells from peripheral blood mononuclear cells, and capable of culturing them in high purity and in large quantities. And a therapeutic / prophylactic agent containing the ⁇ ⁇ cells obtained by the method in immunotherapy for cancer patients, infectious diseases, etc. With the goal.
  • the concentration of a bisphosphonate bone metabolizer (hereinafter abbreviated as bisphosphonate) in peripheral blood mononuclear cells is 0.05 to 100 / ⁇ and interleukin 2 (hereinafter IL— 2) (abbreviated as 2) is added so that the concentration is 50 to 20000 U / mL and cultured.
  • bisphosphonate a bisphosphonate bone metabolizer
  • IL— 2 interleukin 2
  • bisphosphonate is added to peripheral blood mononuclear cells in the culture solution so as to have a concentration of 0.05 to 100 M.
  • ⁇ ⁇ cells selectively activated and expanded or proliferated and further stimulated by adding IL-12 to 50-2000 U / mL. It can be obtained in high purity and in large quantities. Also got The T 6 ⁇ cells contain at least 40% of cells expressing CD 56.
  • the bisphosphonate is added at the start of culture.
  • ⁇ cells obtained by the present invention have a non-specific cytotoxic function, a cancer antigen-specific cytotoxic function, or both cytotoxic functions. Therefore, it can be used as a therapeutic / preventive agent for cancer diseases and infectious diseases.
  • the ⁇ 5 ⁇ cell obtained by the present invention contains a large amount of CD 56-positive ⁇ ⁇ cells having a high cytotoxic function. Therefore, by selecting for CD56-positive cells, it is possible to obtain a higher therapeutic / prophylactic effect when the same number of cells are administered.
  • the therapeutic / prophylactic agent prepared by the present invention has a non-specific cytotoxic function, a cancer antigen-specific cytotoxic function, or both cytotoxic functions.
  • High content of r ⁇ and high cytotoxic function among r ⁇ ⁇ cells Because it contains a lot of 5 T cells, it can identify cancer cells or infected cells without attacking normal cells. It is effective in the treatment of various cancer types and infectious diseases, especially when ⁇ ⁇ cells are derived from autologous lymphocytes and are administered to the patient's own immunity. It is possible to perform its function without being excluded by the system.
  • the concentration of the bisphosphonate bone metabolizing agent in the peripheral blood mononuclear cells is from 0.05 to ⁇ 10 1/1 and the concentration of IL-1 is from 50 to It is characterized by selectively activating and / or proliferating ⁇ cells by adding and culturing at 200 U / mL to obtain a cell population containing ⁇ 5 ⁇ cells with high purity. To do.
  • Bisphosphonates may have any action to suppress bone resorption and are generally used as a therapeutic agent for osteoporosis.
  • Examples thereof include pamidronic acid, a salt thereof and / or a hydrate thereof (eg, pamidronic acid Disodium pentahydrate (A redia, Novartis Pharma), alendronate, its salts and / or their hydrates (eg, sodium alendronate trihydrate (O ne 1 ast, ⁇ ⁇ ) Pharmaceutical)), zoledronic acid, its salts and / or their hydrates (for example, zole Sodium Dronate Hydrate (Zome ta, Novartis Pharma), Risedronic acid, its salts and Z or their hydrates (eg, risedronate sodium 'hydrate), ibandronic acid, its salts and Z or a hydrate thereof (eg, ibandronate disodium), incadronic acid, a salt thereof and Z or a hydrate thereof (eg, disodium incadronate),
  • peripheral blood by collecting blood.
  • the appropriate amount is 15-25 mL. If this amount can be obtained, it can be suitably cultured. However, the range is not limited to this range as long as the peripheral blood volume is sufficient at the start of culture, and the upper limit may be further increased if the burden on the donor to collect blood is small.
  • peripheral blood mononuclear cells by, for example, density gradient centrifugation. About 10 to 7 peripheral blood mononuclear cells can be obtained from 15 to 25 mL of peripheral blood.
  • a suspension of peripheral blood mononuclear cells is referred to as a cell suspension.
  • the culture medium shown here it can be used for culturing cells such as RPMI-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen, hereinafter referred to as DMEM), Iskov medium (Invitrogen, hereinafter referred to as IMEM), etc. You may use the commercially available culture solution used.
  • serum may be added at 0.1 to 20%.
  • serum for example, fetal calf serum (Feta1CalfSerum, hereinafter referred to as FCS), AB serum, autologous plasma, or the like may be used.
  • IL-2 to the above culture solution at a concentration of 50 to 2000 UZmL, more preferably. Preferably, add 400 to 100 OUZmL.
  • 6) with IL- 2 34 to 38 ° C were added thereto, and more preferably at 37, 2-10%, and more preferably are cultured at 5% C_ ⁇ 2 presence.
  • a culture solution is appropriately added according to the number of cells to be cultured.
  • the IL-12 concentration is appropriately added so as to be 50 to 200 OU / mL, more preferably 400 to LOO OUZmL.
  • the culture period is 7 days or longer, a cell group containing ⁇ cells with high purity can be obtained. However, in order to further increase the number of r ⁇ cells, it is preferable to culture for about 14 days.
  • the ⁇ ⁇ cells obtained in this way contain a lot of CD 56 positive ⁇ ⁇ ⁇ cells with high cytotoxic function.
  • CD 56 positive ⁇ ⁇ ⁇ cells can also be selected and used. Is possible. Examples of methods for sorting CD 56-positive ⁇ cells include magnetic cell separation (Magnetic Cell Sorting, hereinafter referred to as MAC S) and flow cytometry. .
  • Magnetic Cell Sorting Magnetic Cell Sorting
  • ⁇ T cells can be efficiently proliferated.
  • the cells obtained by the culture method of the present invention are collected by centrifugation or the like.
  • the washing solution is preferably an isotonic solution having an osmotic pressure equal to that of cells, and more preferably a liquid that can be used as a pharmaceutical product.
  • physiological saline PBS ( ⁇ hosphatebuferfelineseline; phosphate buffered saline), or the like.
  • the r 3 ⁇ cells obtained after washing are collected using a centrifugal method, etc., and suspended in a liquid that can be used as a medicine, such as physiological saline, to prepare the therapeutic / preventive agent of the present invention. can do.
  • a liquid that can be used as a medicine such as physiological saline
  • the amount of the suspension liquid used is appropriately adjusted according to the number of cells to be administered and the administration method.
  • the number of r (5 ⁇ cells used in the therapeutic / prophylactic agent of the present invention is appropriately selected according to the administration method, the type of disease, the symptom of the patient, etc., but is usually 10 8 to 10 12 Z. More preferably, it is 10 9 or more.
  • cytokines such as 1 to 2 and 1 to 1 2
  • the prevention and treatment agent of the present invention it is also possible to combine cytokines such as 1 to 2 and 1 to 1 2 with the prevention and treatment agent of the present invention.
  • interferon interferon
  • prophylactic / therapeutic agent when used as a therapeutic / preventive agent for viral infections, it is possible to combine the interferon (IFN-r) and the like with the prophylactic / therapeutic agent of the present invention.
  • a method of administration for example, it may be injected intravenously, intradermally, subcutaneously, etc., may be directly injected into the affected area, or may be administered systemically as an instillation. Furthermore, it may be injected from an artery near the lesion.
  • the therapeutic / prophylactic agent of the present invention has a high content of T ⁇ 5 T cells having non-specific cytotoxic function, cancer antigen-specific cytotoxic function or both cytotoxic functions, In particular, it contains many CD 56 positive r ⁇ ⁇ cells with high cytotoxic function, so it has the ability to specifically attack cancer cells or infected cells without attacking normal cells. It is an effective treatment and prevention agent for the treatment of cancer types and infectious diseases.
  • the ⁇ 5 cells are derived from autologous lymphocytes, they can function without being excluded by the patient's own immune system when administered to the patient.
  • Example 1
  • AIM-V a culture solution AIM-V (hereinafter referred to as AIM-V (10 FCS)) to which FCS was added at 10%.
  • I L-2 was added at a concentration of 400,
  • the culture solution AIMV was added and cultured for 14 days.
  • the percentage of cells expressing ⁇ 5 T cells in the cell group obtained after 14 days of culture was determined as the fluorescence—Ac tivated C It was measured by ell Sorter (hereinafter referred to as FAC S, Epics XL-MCL ADC, Beckman Cole Yuichi). The values obtained by measurement are shown in Tables 1 to 3 below.
  • the concentration of bisphosphonate bone metabolism-improving drug should be 0 ⁇ 1 to 30 ⁇ , and IL-1 should be added to 400 to 200 OUZmL. It was confirmed that it was preferable. Further, it was confirmed that Ar ed i a is preferably added at a concentration of 1 to 30 ⁇ , On c l a st at a concentration of 1 to 30 / iM, and Zome t a at a concentration of 0.1 to 10 M. Table 1:% of T cells contained in mononuclear cells in which the concentration of IL-12 was changed when Ar e dia was used as the bisphosphonate (%)
  • Peripheral blood was collected from healthy individuals, and peripheral blood mononuclear cells were separated using a specific gravity solution for blood cell separation.
  • peripheral blood mononuclear cells obtained in 1) were suspended in the culture medium AIMV (10% FCS).
  • Ardia was added to the peripheral blood mononuclear cell suspension to a concentration of 10 M.
  • IL 1 was added so that the concentration of IL-1 was 20 00, 40 0, 700, 1 000 and 1 50 OUZmL.
  • Table 4 and Table 5 show the percentage (%) of 5 ⁇ cells when the amount of IL-2 is changed.
  • Table 4 shows the number of cells on days 7, 9, 1 and 14 and Table 5 shows the number of cells on days 0, 3, 7, 9, 1 and 14 (X 1 0 6 ) respectively.
  • peripheral blood mononuclear cells in Table 4 a) and Table 5 a) are derived from the same donor.
  • the peripheral blood mononuclear cells in Tables 4b) and 5b) are from the same donor. From these results, it was confirmed that the amount of IL-12 is preferably 400-1500 UZmL and the number of culture days is 11 days or more in order to obtain high purity and large amount of ⁇ cells.
  • Table 4 A ratio of ⁇ 5 T cells when the amount of IL-12 is changed ()
  • peripheral blood mononuclear cells were separated using a specific gravity solution for blood cell separation.
  • IL-2 was added to a concentration of 700 U / mL. Furthermore, according to the proliferation of the cells, the culture solution A I M-V was added and cultured for 14 days.
  • ⁇ cells activated and / or expanded by adding A redia to peripheral blood mononuclear cells express NKG2D.
  • ⁇ KG 2 D is an active NK cell receptor that is expressed on NK cells, ⁇ 6 T cells and CD 8 positive T cells and expresses its ligand, MI CAZB molecule.
  • Cells are known to be injured through NKG 2DZM ICA interaction.
  • EZT ratio ratio of reaction cells to target cells
  • An n + indicates the number of cells that have developed the fluorescence of Anne x i n V, and An n ⁇ indicates the number of cells that have not developed color.
  • 7 AAD + indicates the number of cells that developed 7-AAD fluorescence, and 7 AAD- indicates the number of cells that did not. That is, An n-7 AAD + is a necrotic cell, An n + 7 AAD + is a late apoptotic cell, An n + 7 AAD- is an early apoptotic cell, Ann-7 AAD- Indicates that the cell is alive.
  • CD 56-positive ⁇ ⁇ cells have higher cytotoxic activity against U 2 66 than CD 56-negative ⁇ ⁇ cells.
  • the expression of NKG 2 D was also confirmed in CD 56 negative ⁇ ⁇ cells, confirming that ⁇ ⁇ cells expressing both NKG2D and CD 56 have more efficient cytotoxic activity. It was.

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Abstract

Cette invention vise à fournir une méthode pour la culture des cellules ϜδT, ces cellules ϜδT des monocytes du sang périphérique pouvant être activées de manière sélective et/ou proliférées et cultivées en grande quantité et à haut degré de pureté; des cellules ϜδT cultivées grâce à cette méthode ; et un remède/traitement préventif contenant des cellules ϜδT cultivées grâce à cette méthode à utiliser dans l'immunothérapie ciblant les patients cancéreux ou atteints d'infections. En ajoutant entre 0,05 et 100 µM de bisphosphonate aux monocytes du sang périphérique, les cellules ϜδT dans les monocytes du sang périphérique sont activées de manière sélective et/ou proliférées et ensuite activées et/ou proliférées en les stimulant avec 50 à 2000 U/mL d’IL-2. Par conséquent, les cellules ϜδT peuvent être cultivées en grande quantité et à haut degré de pureté. Les cellules ϜδT ainsi obtenues contiennent une grande quantité de cellules CD56 positives ayant une activité cytotoxique élevée, ce qui les rend utiles en tant que remède ou traitement préventif chez les patients atteints de cancer ou d’une infection.
PCT/JP2005/013218 2004-07-13 2005-07-12 MÉTHODE POUR FAIRE LA CULTURE DES CELLULES ϜδT, LES CELLULES ϜδT ET UN REMÈDE/TRAITEMENT PRÉVENTIF WO2006006720A1 (fr)

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JP2006529212A JPWO2006006720A1 (ja) 2004-07-13 2005-07-12 γδＴ細胞の培養方法、γδＴ細胞及び治療・予防剤

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Cited By (18)

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WO2008152822A1 (fr) * 2007-06-15 2008-12-18 Medinet Co., Ltd. Agent médicinal
WO2010001599A1 (fr) 2008-07-01 2010-01-07 株式会社メディネット Procédé d'induction simultanée de cellules ctl et γδt
JPWO2008111430A1 (ja) * 2007-03-09 2010-06-24 タカラバイオ株式会社 γδＴ細胞集団の製造方法
US7749760B2 (en) 2008-07-10 2010-07-06 Hyogo College Of Medicine Vγ9Vδ2 T cell proliferation agent, method for producing activated Vγ9Vδ2 T cells, and uses thereof
WO2011096504A1 (fr) * 2010-02-08 2011-08-11 株式会社日本バイオセラピー研究所 Procédé de production d'un produit sanguin du type à amplification des cellules nk
WO2012099093A1 (fr) * 2011-01-21 2012-07-26 株式会社日本バイオセラピー研究所 Procédé pour la production de préparation du sang enrichi en cellules nk
JP2013081428A (ja) * 2011-10-11 2013-05-09 Biotherapy Institute Of Japan Ｃｄ５６陽性ｔ細胞増強方法
CN104293734A (zh) * 2014-09-28 2015-01-21 上海云舜生物技术有限公司 一种人γδT细胞的制备方法
CN104651308A (zh) * 2013-11-22 2015-05-27 深圳先进技术研究院 一种用磷酸盐扩增γδT细胞的方法及其应用
WO2016005752A1 (fr) 2014-07-09 2016-01-14 Tc Biopharm Ltd Lymphocytes t gamma delta et leurs utilisations
WO2016060111A1 (fr) * 2014-10-14 2016-04-21 学校法人 聖マリアンナ医科大学 Procédé de production de cellules t gamma-delta, et produit pharmaceutique
WO2016166544A1 (fr) 2015-04-15 2016-10-20 Tc Biopharm Ltd Cellules gamma delta modifiées et leurs utilisations
CN106795493A (zh) * 2014-10-06 2017-05-31 堤乐哈修门医学研究基础建设及服务有限公司 使用双膦酸盐、抗cd3抗体和il‑2扩增t细胞群体
WO2018143243A1 (fr) * 2017-02-03 2018-08-09 国立大学法人神戸大学 Procédé de production de cellules souches pluripotentes induites
WO2020013315A1 (fr) 2018-07-13 2020-01-16 国立大学法人京都大学 PROCÉDÉ DE PRODUCTION DE LYMPHOCYTES T γδ
JP2020506713A (ja) * 2017-02-07 2020-03-05 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ 多能性幹細胞から模倣自然免疫細胞を生成する方法及びキット
WO2021085504A1 (fr) 2019-11-01 2021-05-06 京都府公立大学法人 Récepteur d'anticorps de lympocyte b et son utilisation
CN118703437A (zh) * 2024-08-27 2024-09-27 上海吉泰依科赛生物科技有限公司 一种γδT细胞无血清培养基及其应用

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013176403A (ja) * 2007-03-09 2013-09-09 Takara Bio Inc γδＴ細胞集団の製造方法
JPWO2008111430A1 (ja) * 2007-03-09 2010-06-24 タカラバイオ株式会社 γδＴ細胞集団の製造方法
WO2008152822A1 (fr) * 2007-06-15 2008-12-18 Medinet Co., Ltd. Agent médicinal
JPWO2008152822A1 (ja) * 2007-06-15 2010-08-26 株式会社メディネット 医薬
WO2010001599A1 (fr) 2008-07-01 2010-01-07 株式会社メディネット Procédé d'induction simultanée de cellules ctl et γδt
US8962313B2 (en) 2008-07-01 2015-02-24 Medinet Co., Ltd. Method for the simultaneous induction of CTL and γδT cell
CN102137925B (zh) * 2008-07-01 2014-12-10 迈世耐特股份公司 同时诱导CTL和γδT细胞的方法
JP5524056B2 (ja) * 2008-07-01 2014-06-18 株式会社メディネット ＣＴＬとγδＴ細胞の同時誘導方法
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