JP2530966B2 - High-purity mass culture method for functional T cell subgroup - Google Patents
High-purity mass culture method for functional T cell subgroupInfo
- Publication number
- JP2530966B2 JP2530966B2 JP4054433A JP5443392A JP2530966B2 JP 2530966 B2 JP2530966 B2 JP 2530966B2 JP 4054433 A JP4054433 A JP 4054433A JP 5443392 A JP5443392 A JP 5443392A JP 2530966 B2 JP2530966 B2 JP 2530966B2
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- Prior art keywords
- cells
- antibody
- cell
- positive
- functional
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims description 83
- 238000012136 culture method Methods 0.000 title claims description 5
- 238000000034 method Methods 0.000 claims description 39
- 238000012258 culturing Methods 0.000 claims description 12
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 claims description 5
- 229930192851 perforin Natural products 0.000 claims description 5
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 230000004936 stimulating effect Effects 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 75
- 102000000588 Interleukin-2 Human genes 0.000 description 16
- 108010002350 Interleukin-2 Proteins 0.000 description 16
- 238000000926 separation method Methods 0.000 description 14
- 238000001179 sorption measurement Methods 0.000 description 14
- 230000000259 anti-tumor effect Effects 0.000 description 13
- 210000005259 peripheral blood Anatomy 0.000 description 11
- 239000011886 peripheral blood Substances 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 239000000427 antigen Substances 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- HIYAVKIYRIFSCZ-CYEMHPAKSA-N 5-(methylamino)-2-[[(2S,3R,5R,6S,8R,9R)-3,5,9-trimethyl-2-[(2S)-1-oxo-1-(1H-pyrrol-2-yl)propan-2-yl]-1,7-dioxaspiro[5.5]undecan-8-yl]methyl]-1,3-benzoxazole-4-carboxylic acid Chemical compound O=C([C@@H](C)[C@H]1O[C@@]2([C@@H](C[C@H]1C)C)O[C@@H]([C@@H](CC2)C)CC=1OC2=CC=C(C(=C2N=1)C(O)=O)NC)C1=CC=CN1 HIYAVKIYRIFSCZ-CYEMHPAKSA-N 0.000 description 4
- HIYAVKIYRIFSCZ-UHFFFAOYSA-N calcium ionophore A23187 Natural products N=1C2=C(C(O)=O)C(NC)=CC=C2OC=1CC(C(CC1)C)OC1(C(CC1C)C)OC1C(C)C(=O)C1=CC=CN1 HIYAVKIYRIFSCZ-UHFFFAOYSA-N 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- 238000009169 immunotherapy Methods 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 4
- 241001494479 Pecora Species 0.000 description 3
- 108091008874 T cell receptors Proteins 0.000 description 3
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
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- 239000012894 fetal calf serum Substances 0.000 description 2
- 238000010230 functional analysis Methods 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 210000003359 CD4-positive helper T lymphocyte Anatomy 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- XECAHXYUAAWDEL-UHFFFAOYSA-N acrylonitrile butadiene styrene Chemical compound C=CC=C.C=CC#N.C=CC1=CC=CC=C1 XECAHXYUAAWDEL-UHFFFAOYSA-N 0.000 description 1
- 239000004676 acrylonitrile butadiene styrene Substances 0.000 description 1
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
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- 239000001913 cellulose Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
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- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、目的とする機能的Ｔ細
胞亜群が少量しか含まれない血液、骨髄等のソ−スから
該機能的Ｔ細胞亜群を高純度にかつ大量に増殖培養する
方法および当該方法により得られた機能的Ｔ細胞に関す
るものである。FIELD OF THE INVENTION The present invention relates to the proliferation of a large amount of a target functional T cell subgroup with high purity from a source such as blood or bone marrow containing a small amount of the target functional T cell subgroup. The present invention relates to a method for culturing and a functional T cell obtained by the method.
【０００２】[0002]
【従来の技術及び発明が解決しようとする課題】従来、
例えば、癌の免疫療法の一つである養子免疫療法では、
大量の白血球を含む癌患者末梢血等を採取し、該血液よ
りＴ細胞亜集団を比重液にて分離し、インタ−ロイキン
２（以下ＩＬ−２という）と共に培養し、癌細胞破壊機
能を持つＴ細胞を活性化し、再び、患者に戻す。当該方
法は、大量の白血球を採取する必要があり、該作業は患
者に大きな負担をかけるものであった。更に、従来の比
重液を使用する分離方法ではＴ細胞のみを分離すること
はできなかった。また更には、当該方法で分離したＴ細
胞は亜集団として分離されているため、様々な機能を持
つＴ細胞が混在している。このため、癌細胞破壊に対し
抑制的に働くＴ細胞をも活性化させる可能性があった。2. Description of the Related Art
For example, in adoptive immunotherapy, which is one of cancer immunotherapy,
Peripheral blood of a cancer patient containing a large amount of white blood cells is collected, a T cell subpopulation is separated from the blood with a specific gravity liquid, and cultured with interleukin 2 (hereinafter referred to as IL-2) to have a cancer cell-disrupting function. T cells are activated and returned to the patient. This method requires a large amount of white blood cells to be collected, and this work places a heavy burden on the patient. Furthermore, it was not possible to separate only T cells by the conventional separation method using a specific gravity liquid. Furthermore, since the T cells separated by the method are separated as a subpopulation, T cells having various functions are mixed. Therefore, there is a possibility of activating T cells that suppressively act on the destruction of cancer cells.
【０００３】癌患者の負担を軽減し、Ｔ細胞の亜集団の
みを分離する方法として、少量の患者末梢血等を採取
し、比重液を用いてＴ細胞亜集団を採取し、該Ｔ細胞亜
集団を抗ＣＤ３抗体固相化プレ−ト中で刺激後培養する
ことで大量のＴ細胞亜集団のみを培養することが可能と
なった。しかし、該方法においても前記の方法と同様癌
細胞破壊に対し抑制的に働くＴ細胞をも活性化させる可
能性があった。As a method for reducing the burden on a cancer patient and separating only a subpopulation of T cells, a small amount of peripheral blood of a patient is collected, and a T cell subpopulation is collected by using a specific gravity liquid. By culturing the population after stimulation in an anti-CD3 antibody-immobilized plate, it became possible to culture only a large amount of T cell subpopulation. However, even in this method, there is a possibility that T cells that act suppressively against the destruction of cancer cells may be activated as in the above method.
【０００４】このため、我々は、鋭意検討を重ねた結
果、目的の機能的Ｔ細胞亜群が少量しか含まれないソ−
スから、該機能的Ｔ細胞亜群を高純度にかつ大量に増殖
培養する方法および当該方法で得られる機能的Ｔ細胞亜
群を発明するに至った。[0004] Therefore, as a result of extensive studies, we have found that the target functional T cell subgroup is contained in a small amount.
Have invented a method for proliferating and culturing the functional T cell subgroup with high purity and in a large amount, and a functional T cell subgroup obtained by the method.
【０００５】本発明により得られる機能的Ｔ細胞亜群
は、前記の養子免疫療法に限らず、Ｔ細胞抗原の基礎的
解析の研究等広く応用できるものである。The functional T cell subgroup obtained by the present invention is not limited to the above-mentioned adoptive immunotherapy, but can be widely applied to studies of basic analysis of T cell antigens.
【０００６】[0006]
【課題を解決するための手段】上記課題を解決するため
の本発明の手段は、目的の抗Ｔ細胞抗原抗体に反応する
Ｔ細胞亜群を分離させ、抗ＣＤ３抗体固相化プレ−ト中
で刺激させたのち増殖培養することを特徴とする機能的
Ｔ細胞亜群の高純度大量培養方法、および、当該方法で
得られる機能的Ｔ細胞亜群を提供することである。Means for Solving the Problems The means of the present invention for solving the above-mentioned problems are to separate T cell subgroups that react with an anti-T cell antigen antibody of interest, and to prepare an anti-CD3 antibody-immobilized plate. The present invention provides a method for high-purity large-scale culturing of a functional T cell subgroup, which comprises stimulating the cells with the above method and then proliferating and culturing, and a functional T cell subgroup obtained by the method.
【０００７】機能的Ｔ細胞亜群を分離する方法として
は、セルソ−タ−を用いる方法、吸着分離材を用いる方
法等がある。Methods for separating functional T cell subgroups include a method using a cell sorter and a method using an adsorption separation material.
【０００８】前記吸着分離材とは、担体に目的とするＴ
細胞上の抗原と特異的に反応する抗Ｔ細胞抗原抗体を担
持させたものを用いることができる。The above-mentioned adsorption / separation material is the target T
What carried the anti- T cell antigen antibody which reacts specifically with the antigen on a cell can be used.
【０００９】前記担体としては、例えばキチン・キトサ
ン、セルロ−ス、アガロ−ス、ポリビニルアルコ−ル、
ポリメチルメタアクリレ−ト、ポリスチレン、アクリロ
ニトリル・ブタジエン・スチレン樹脂、ガラス、シリカ
等を用いることができる。また、市販されている吸着用
ビ−ズを用いてもよい。更には、吸着分離後抗ＣＤ３抗
体固相化プレ−ト中で刺激するため吸着分離材は目的と
するＴ細胞よりも小さいものが望ましい。Examples of the carrier include chitin / chitosan, cellulose, agarose, polyvinyl alcohol,
Polymethylmethacrylate, polystyrene, acrylonitrile-butadiene-styrene resin, glass, silica and the like can be used. Alternatively, commercially available adsorption beads may be used. Furthermore, since it is stimulated in the anti-CD3 antibody-immobilized plate after adsorption separation, the adsorption separation material is preferably smaller than the target T cell.
【００１０】前記担体に担持させる抗Ｔ細胞抗原抗体と
しては、例えば抗ＣＤ１ａ抗体、抗ＣＤ１ｂ抗体、抗Ｃ
Ｄ１ｃ抗体、抗ＣＤ２抗体、抗ＣＤ２Ｒ抗体、抗ＣＤ４
抗体、抗ＣＤ５抗体、抗ＣＤ６抗体、抗ＣＤ７抗体、抗
ＣＤ８抗体、抗ＣＤ２７抗体、抗ＣＤ２８抗体、抗ＣＤ
２９抗体、抗ＣＤｗ６０抗体、抗γδ抗体等目的とする
Ｔ細胞抗原に反応する抗体であればよい。Examples of the anti-T cell antigen antibody carried on the carrier include anti-CD1a antibody, anti-CD1b antibody and anti-C antibody.
D1c antibody, anti-CD2 antibody, anti-CD2R antibody, anti-CD4
Antibody, anti-CD5 antibody, anti-CD6 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD
Any antibody such as 29 antibody, anti-CDw60 antibody, anti-γδ antibody, etc. that reacts with the target T cell antigen may be used.
【００１１】実施例 次に本発明の実施例について具体的に説明する。EXAMPLES Next, examples of the present invention will be specifically described.
【００１２】実施例１ 健常者より末梢血を２０ｍｌ採血し、比重液によりリン
パ球層を分離し、次にナイロンウ−ルを通し、単球およ
びＢ細胞を除去したＴ細胞高純度液を得た。フィコエリ
シンでラベリングした抗ＣＤ４抗体を用意し、該Ｔ細胞
高純度液を反応させた後セルソ−タ−（ＦＡＣＳｔｅ
ｒ：ベクトンデッキンソン社製）にて該抗ＣＤ４抗体と
反応したＴ細胞を１×１０6 個分離した。 Example 1 20 ml of peripheral blood was collected from a healthy person, the lymphocyte layer was separated by a specific gravity solution, and then nylon wool was passed through to obtain a highly purified T cell liquid free of monocytes and B cells. . An anti-CD4 antibody labeled with phycoerythin was prepared and reacted with the T cell high-purity liquid, and then the cell sorter (FACSte).
r: Becton Dickinson) was used to separate 1 × 10 6 T cells that reacted with the anti-CD4 antibody.
【００１３】あらかじめ、抗ＣＤ３抗体をプレ−トに固
相化させた抗ＣＤ３抗体固相化プレ−トを準備してお
き、該プレ−トに上記抗ＣＤ４抗体反応Ｔ細胞を一昼夜
刺激培養した。An anti-CD3 antibody-immobilized plate having an anti-CD3 antibody immobilized on a plate was prepared in advance, and the anti-CD4 antibody-reactive T cells were stimulated and cultured in the plate overnight. .
【００１４】該ＣＤ３刺激Ｔ細胞をプレ−トを用いて３
７℃でＩＬ−２および牛胎児血清を含むＲＰＭＩ１６４
０培地で３５日間培養を行った。培養は１×１０6 個／
ｍｌに調整し培養を開始し、７日後に増殖した細胞密度
を測定し、再度１×１０6 個／ｍｌに調整し継代を行っ
た。The CD3-stimulated T cells were plated with a plate 3
RPMI164 containing IL-2 and fetal calf serum at 7 ° C
Culture was carried out in 0 medium for 35 days. Culture is 1 × 10 6 cells /
The cells were adjusted to ml and the culture was started, and after 7 days, the density of the cells that had proliferated was measured, adjusted again to 1 × 10 6 cells / ml, and passaged.
【００１５】上記の方法による細胞密度および該細胞の
ＣＤ４陽性率の結果を表１に示す。Table 1 shows the results of the cell density and the CD4 positive rate of the cells obtained by the above method.
【００１６】[0016]
【表１】
[Table 1]
【００１７】培養期間を通じてＣＤ４陽性率は９９．２
％以上を示した。今回の実験は培養７日目で継代を行っ
たが、全ての該細胞を培養した場合、培養開始時１×１
０6個の細胞が、計算上３５日間で１．１×１０14個に
増殖することになる。The CD4 positive rate was 99.2 throughout the culture period.
% Or more. In this experiment, the cells were passaged on the 7th day of culture, but when all the cells were cultured, 1 × 1 at the start of culture
A total of 0 6 cells will grow to 1.1 x 10 14 in 35 days.
【００１８】実施例２ 実施例１と同様に健常者より末梢血２０ｍｌを採血し、
Ｔ細胞高純度液を得た。フィコエリシンでラベリングし
た抗ＣＤ８抗体を用意し、該抗ＣＤ８抗体と該Ｔ細胞高
純度液を反応させた後セルソ−タ−（ＦＡＣＳｔｅｒ：
ベクトンデッキンソン社製）にて該抗ＣＤ８抗体と反応
したＴ細胞を１×１０6 個分離した。 Example 2 As in Example 1, 20 ml of peripheral blood was collected from a healthy person,
A highly purified T cell solution was obtained. An anti-CD8 antibody labeled with phycoerythin was prepared, and the anti-CD8 antibody was allowed to react with the highly purified T cell solution, and then a cell sorter (FACSter:
1 × 10 6 T cells reacted with the anti-CD8 antibody were separated by Becton Dickinson).
【００１９】あらかじめ、抗ＣＤ３抗体をプレ−トに固
相化させた抗ＣＤ３抗体固相化プレ−トを準備してお
き、該プレ−トに上記該抗ＣＤ８抗体反応Ｔ細胞を一昼
夜刺激培養した。An anti-CD3 antibody-immobilized plate having an anti-CD3 antibody immobilized on a plate is prepared in advance, and the anti-CD8 antibody-reactive T cells are stimulated in the plate overnight and cultured. did.
【００２０】該ＣＤ３刺激Ｔ細胞をプレ−トを用いて３
７℃でＩＬ−２および牛胎児血清を含むＲＰＭＩ１６４
０培地で３５日間培養を行った。培養は１×１０6 個／
ｍｌに調整し培養を開始し、７日後に増殖した細胞密度
を測定し、再度１×１０6 個／ｍｌに調整し継代を行っ
た。The CD3-stimulated T cells were plated with a plate 3
RPMI164 containing IL-2 and fetal calf serum at 7 ° C
Culture was carried out in 0 medium for 35 days. Culture is 1 × 10 6 cells /
The cells were adjusted to ml and the culture was started, and after 7 days, the density of the cells that had proliferated was measured, adjusted again to 1 × 10 6 cells / ml, and passaged.
【００２１】上記の方法による細胞密度および該細胞の
ＣＤ８陽性率の結果を表２に示す。Table 2 shows the results of the cell density and the CD8 positive rate of the cells obtained by the above method.
【００２２】[0022]
【表２】 [Table 2]
【００２３】培養期間を通じてＣＤ８陽性率は９９．５
％以上を示した。今回の実験は培養７日目で継代を行っ
たが、全ての該細胞を培養した場合、培養開始時１×１
０6個の細胞が、計算上３５日間で４．５×１０13個に
増殖することになる。The CD8 positive rate was 99.5 throughout the culture period.
% Or more. In this experiment, the cells were passaged on the 7th day of culture, but when all the cells were cultured, 1 × 1 at the start of culture
0 6 cells will grow to 4.5 × 10 13 cells in 35 days calculated.
【００２４】実施例３ 市販のダイナビ−ズＭ−４５０ Ｓｈｅｅｐ ａｎｔｉ
- ＭｏｕｓｅＩｇＧ（ＤＹＮＡＬ社）に抗ＣＤ４抗体を
担持させた吸着分離材を準備しておく。 Example 3 Commercially available Dinaviz M-450 Sheep anti
-Prepare an adsorption / separation material in which Mouse IgG (DYNAL) carries an anti-CD4 antibody.
【００２５】実施例１と同様の方法で癌患者４人（乳癌
２人、大腸癌２人）より末梢血２０ｍｌを採血し、Ｔ細
胞高純度液を得た。該Ｔ細胞高純度液と上記抗ＣＤ４抗
体担持吸着分離材を反応させ、ＣＤ４陽性Ｔ細胞を分離
した。In the same manner as in Example 1, 20 ml of peripheral blood was collected from 4 cancer patients (2 breast cancer and 2 colon cancer) to obtain a highly purified T cell liquid. The T cell high-purity liquid was reacted with the anti-CD4 antibody-supporting adsorption separation material to separate CD4-positive T cells.
【００２６】あらかじめ、抗ＣＤ３抗体をプレ−トに固
相化させた抗ＣＤ３抗体固相化プレ−トを準備してお
き、該プレ−トに上記の吸着分離したＣＤ４陽性Ｔ細胞
を一昼夜刺激培養した。An anti-CD3 antibody-immobilized plate in which an anti-CD3 antibody was immobilized in a plate was prepared in advance, and the above-described adsorbed and separated CD4-positive T cells were stimulated overnight in the plate. Cultured.
【００２７】抗ＣＤ３抗体固相化プレ−トで刺激後Ｔ細
胞を取り出し、細胞数を確認した。細胞数は１〜２×１
０6 個であった。該細胞をプレ−トでＩＬ−２と共に７
日間培養を行い、細胞数が２×１０8 個に達したことを
確認後、濃縮回転培養法にて９日間培養を行った。After stimulation with an anti-CD3 antibody-immobilized plate, T cells were taken out and the number of cells was confirmed. The number of cells is 1-2 x 1
0 was six. The cells were plated at 7 with IL-2.
After culturing for 9 days and confirming that the number of cells reached 2 × 10 8 , the cells were cultivated for 9 days by the concentrated rotary culture method.
【００２８】末梢血２０ｍｌから１６日間の培養で得ら
れた細胞数と、該細胞のＣＤ４陽性率の結果を表３に示
す。Table 3 shows the number of cells obtained by culturing from 20 ml of peripheral blood for 16 days and the CD4 positive rate of the cells.
【００２９】[0029]
【表３】 [Table 3]
【００３０】本方法によりＣＤ４陽性Ｔ細胞が純度９５
％以上で、しかも、培養後１６日間で吸着分離後に得ら
れたリンパ球数の少なくとも１６００倍以上の３．２×
１０9 個以上のＣＤ４陽性Ｔ細胞を得ることができた。According to this method, CD4 positive T cells have a purity of 95.
%, And at least 1600 times more than the number of lymphocytes obtained after adsorption and separation in 16 days after culturing 3.2 ×
It was possible to obtain 10 9 or more CD4-positive T cells.
【００３１】一般的に、長期に渡り維持されたＣＤ４陽
性クロ−ンの解析結果では、化学物質であるホルボ−ル
ミリステ−トアセテ−ト（以下ＰＭＡ）とカルシウムイ
オノホアＡ２３１８７（以下Ａ２３１８７）で刺激した
場合、ＩＬ−２の産生能が認められるものの、Ｔ細胞レ
セプタ−を介した刺激ではＩＬ−２産生能が低下する傾
向があると言われている。これは、長期に渡る培養によ
り該刺激に対する反応性が失われていくものと考えられ
る。In general, the results of analysis of CD4-positive clones that were maintained for a long period of time were stimulated by the chemical substances phorbol myristate acetate (PMA) and calcium ionophore A23187 (A23187). In this case, although the IL-2 producing ability is recognized, it is said that the IL-2 producing ability tends to be lowered by stimulation through the T cell receptor. It is considered that the responsiveness to the stimulus is lost due to long-term culture.
【００３２】当該方法で得られた細胞及び長期に渡り維
持された２種類のＣＤ４陽性クロ−ンのＩＬ−２産生能
を測定した。該ＣＤ４陽性クロ−ンはＲａｊｉ細胞を特
異的に認識する細胞であり、２カ月に渡り培養を行った
ものである。当該方法で得られた細胞及び該ＣＤ４陽性
クロ−ンをそれぞれ２×１０6 個／ｍｌに調製し、それ
ぞれの細胞上清及び細胞をＰＭＡ単独、Ａ２３１８７単
独及びＰＭＡとＡ２３１８７で刺激したのちの上清、更
に、当該方法で得られた細胞については抗ＣＤ３抗体、
該ＣＤ４陽性クロ−ンについてはＲａｊｉ細胞で刺激し
たのちの上清中のＩＬ−２濃度を測定した。ＩＬ−２の
単位は塩野義製薬株式会社の単位を用いた。当該方法で
得られた細胞のＩＬ−２産生能の結果を表４に、該ＣＤ
４陽性クロ−ンのＩＬ−２産生能の結果を表５に示す。The IL-2 producing ability of the cells obtained by the method and two types of CD4 positive clones maintained for a long period of time was measured. The CD4 positive clone is a cell that specifically recognizes Raji cells, and has been cultured for 2 months. The cells obtained by the method and the CD4 positive clones were prepared at 2 × 10 6 cells / ml, and the respective cell supernatants and cells were stimulated with PMA alone, A23187 alone and PMA and A23187. And anti-CD3 antibody for cells obtained by the method,
Regarding the CD4 positive clone, the IL-2 concentration in the supernatant after stimulation with Raji cells was measured. As a unit of IL-2, a unit of Shionogi Pharmaceutical Co., Ltd. was used. The results of the IL-2 producing ability of the cells obtained by the method are shown in Table 4 and
Table 5 shows the results of the IL-2 producing ability of 4-positive clones.
【００３３】[0033]
【表４】 [Table 4]
【００３４】[0034]
【表５】 [Table 5]
【００３５】該ＣＤ４陽性クロ−ンはＴ細胞レセプタ−
を介した刺激であるＲａｊｉ細胞での刺激に対しＩＬ−
２の産生能は認められなかった。これに対し、当該方法
で得られた細胞をＴ細胞レセプタ−を介した刺激である
抗ＣＤ３抗体での刺激に対しＩＬ−２産生能が認められ
た。The CD4 positive clone is a T cell receptor.
IL-in response to stimulation in Raji cells, which is mediated by
No productivity of 2 was observed. On the other hand, IL-2 producing ability was observed in the cells obtained by the method, when stimulated with anti-CD3 antibody, which is stimulation through T cell receptor.
【００３６】更に当該方法で得られたＣＤ４陽性Ｔ細胞
を抗パ−フォリン抗体で反応させ免疫染色を行った結
果、５０〜６０％が抗パ−フォリン陽性Ｔ細胞であっ
た。つまり、当該方法で得られれた細胞の５０〜６０％
はＣＤ４陽性かつパ−フォリン陽性細胞であった。Further, the CD4-positive T cells obtained by the above method were reacted with an anti-perforin antibody and immunostained. As a result, 50-60% were anti-perforin-positive T cells. That is, 50 to 60% of the cells obtained by the method
Were CD4 positive and perforin positive cells.
【００３７】パ−フォリン陽性Ｔ細胞はキラ−Ｔ細胞と
呼ばれ、抗腫瘍活性を持つ。このため、当該方法で得ら
れた細胞の樹立細胞への抗腫瘍活性を測定した。該樹立
細胞には、抗ＣＤ３抗体産生細胞であるＯＫＴ−３と、
Ｋ５６２、Ｄａｕｄｉ及びＵ９３７の４種類を用いた。
Ｋ５６２、Ｄａｕｄｉ及びＵ９３７については、抗ＣＤ
３抗体存在下での抗腫瘍活性を測定した。測定は、該細
胞数と該樹立細胞数の割合（以下Ｅ／Ｔ比という）を２
０：１で反応させた。結果を表６に示す。Perforin-positive T cells are called killer T cells and have antitumor activity. Therefore, the antitumor activity of the cells obtained by the method on established cells was measured. The established cells include anti-CD3 antibody-producing cells OKT-3,
Four types of K562, Daudi and U937 were used.
Anti-CD for K562, Daudi and U937
Antitumor activity in the presence of 3 antibodies was measured. For the measurement, the ratio of the number of cells to the number of established cells (hereinafter referred to as E / T ratio) is 2
The reaction was carried out at 0: 1. The results are shown in Table 6.
【００３８】[0038]
【表６】 [Table 6]
【００３９】当該方法で得られた細胞はＯＫＴ−３、Ｋ
５６２、Ｄａｕｄｉ及びＵ９３７に対し高い抗腫瘍活性
を示した。The cells obtained by this method are OKT-3, K
It showed high antitumor activity against 562, Daudi and U937.
【００４０】以上、実験例３により得られた細胞はＣＤ
４陽性率が９５％以上であり、大量培養を行ったにもか
かわらず生理的刺激条件下でＩＬ−２を産生し、更に、
その５０〜６０％がパ−フォリン陽性で高い抗腫瘍活性
が認められた。As described above, the cells obtained in Experimental Example 3 were CD
4-Positive rate is 95% or more, IL-2 is produced under physiological stimulation conditions despite large-scale culture, and further,
50 to 60% of them were perforin positive and high antitumor activity was observed.
【００４１】従来ＣＤ４陽性Ｔ細胞はヘルパ−Ｔ細胞と
呼ばれている。一方抗腫瘍活性を持つパ−フォリン陽性
細胞はキラ−細胞と呼ばれている。当該方法で得られた
ＣＤ４陽性細胞はヘルパ−とキラ−の両者の機能を備え
るＣＤ４陽性ヘルパ−／キラ−Ｔ細胞である。当該方法
で得られる細胞はＴ細胞抗原の機能解析の研究を進める
上でも大きな意味を持つものであり、癌の養子免疫療法
へも大いに期待されるものである。Conventionally, CD4-positive T cells are called helper T cells. On the other hand, perforin-positive cells having antitumor activity are called killer cells. The CD4-positive cells obtained by the method are CD4-positive helper / killer T cells having both the helper and killer functions. The cells obtained by this method have great significance in advancing research into the functional analysis of T cell antigens, and are also highly expected for adoptive immunotherapy for cancer.
【００４２】実施例４ 市販のダイナビ−ズＭ−４５０ Ｓｈｅｅｐ ａｎｔｉ
- ＭｏｕｓｅＩｇＧ（ＤＹＮＡＬ社）に抗ＣＤ８抗体を
担持させた吸着分離材を準備しておく。 Example 4 Commercially available Dinaviz M-450 Sheep anti
-Prepare an adsorption / separation material in which Mouse IgG (DYNAL) carrying anti-CD8 antibody is prepared.
【００４３】実施例１と同様の方法で癌患者４人（乳癌
２人、大腸癌２人）より末梢血２０ｍｌを採血し、Ｔ細
胞高純度液を得た。該Ｔ細胞高純度液と上記抗ＣＤ８抗
体担持吸着分離材を反応させ、ＣＤ８陽性Ｔ細胞を分離
した。In the same manner as in Example 1, 20 ml of peripheral blood was collected from 4 cancer patients (2 breast cancer, 2 colon cancer) to obtain a highly purified T cell liquid. The highly purified T cell solution was allowed to react with the anti-CD8 antibody-supporting adsorption separation material to separate CD8 positive T cells.
【００４４】あらかじめ、抗ＣＤ３抗体をプレ−トに固
相化させた抗ＣＤ３抗体固相化プレ−トを準備してお
き、該プレ−トに上記の吸着分離したＴ細胞を一昼夜刺
激培養した。An anti-CD3 antibody-immobilized plate in which an anti-CD3 antibody was immobilized in a plate was prepared in advance, and the above-described adsorbed and separated T cells were stimulated and cultured in the plate overnight. .
【００４５】抗ＣＤ３抗体固相化プレ−トで刺激後Ｔ細
胞を取り出し、細胞数を確認した。細胞数は１〜２×１
０6 個であった。該細胞をプレ−トでＩＬ−２と共に７
日間培養を行い、細胞数が２×１０8 個に達したことを
確認後、濃縮回転培養法にて８日間培養を行った。After stimulation with an anti-CD3 antibody-immobilized plate, T cells were taken out and the number of cells was confirmed. The number of cells is 1-2 x 1
0 was six. The cells were plated at 7 with IL-2.
After culturing for a day and confirming that the number of cells reached 2 × 10 8 , the cells were cultivated for 8 days by the concentrated rotary culture method.
【００４６】末梢血２０ｍｌから１５日間の培養で得ら
れた細胞数と、該細胞のＣＤ８陽性率の結果を表７に示
す。Table 7 shows the number of cells obtained by culturing 20 ml of peripheral blood for 15 days and the result of the CD8 positive rate of the cells.
【００４７】[0047]
【表７】 [Table 7]
【００４８】本方法によりＣＤ８陽性Ｔ細胞が純度９
７．３％以上で、しかも、吸着分離後に得られたリンパ
球数の少なくとも２２５０倍以上の４．５×１０9 個以
上のＣＤ８陽性Ｔ細胞を得ることができた。According to this method, CD8-positive T cells have a purity of 9
It was possible to obtain 7.3% or more, and 4.5 × 10 9 or more CD8-positive T cells that were at least 2250 times the number of lymphocytes obtained after adsorption separation.
【００４９】実施例５ 市販のダイナビ−ズＭ−４５０ Ｓｈｅｅｐ ａｎｔｉ
- ＭｏｕｓｅＩｇＧ（ＤＹＮＡＬ社）に抗γδ抗体を担
持させた吸着分離材を準備しておく。 Example 5 Commercially available Dinaviz M-450 Sheep anti
-Prepare an adsorption / separation material in which Mouse IgG (DYNAL) carries an anti-γδ antibody.
【００５０】実施例１と同様に健常者より末梢血２０ｍ
ｌを採血し、Ｔ細胞高純度液を得た。該Ｔ細胞高純度液
と上記抗γδ抗体担持吸着分離材を反応させ、γδ陽性
Ｔ細胞を分離した。As in Example 1, 20 m of peripheral blood from a healthy person
1 was collected to obtain a highly purified T cell liquid. The highly purified T cell solution was reacted with the anti-γδ antibody-supporting adsorption separation material to separate γδ positive T cells.
【００５１】あらかじめ、抗ＣＤ３抗体をプレ−トに固
相化させた抗ＣＤ３抗体固相化プレ−トを準備してお
き、該プレ−トに上記の吸着分離したＴ細胞を一昼夜刺
激培養した。An anti-CD3 antibody-immobilized plate prepared by immobilizing anti-CD3 antibody on a plate was prepared in advance, and the above-described adsorbed and separated T cells were stimulated and cultured on the plate overnight. .
【００５２】抗ＣＤ３抗体固相化プレ−トで刺激後Ｔ細
胞を取り出し、細胞数を確認した。細胞数は１〜２×１
０6 個であった。該細胞をプレ−トでＩＬ−２と共に１
６日間培養を行い、濃縮回転培養法にて４日間培養を行
った。After stimulation with an anti-CD3 antibody-immobilized plate, T cells were taken out and the number of cells was confirmed. The number of cells is 1-2 x 1
0 was six. The cells were plated with IL-2 at 1
Cultivation was carried out for 6 days and then for 4 days by the concentrated rotary culture method.
【００５３】末梢血２０ｍｌから２０日間の培養で得ら
れた細胞数と、該細胞のγδ陽性率の結果を表８に示
す。Table 8 shows the number of cells obtained by culturing from 20 ml of peripheral blood for 20 days and the γδ positive rate of the cells.
【００５４】[0054]
【表８】 [Table 8]
【００５５】当該方法で得られた細胞のＯＫＴ−３、Ｋ
５６２及びＤａｕｄｉの３種類の樹立細胞への抗腫瘍活
性を測定した。Ｋ５６２及びＤａｕｄｉについては抗Ｃ
Ｄ３抗体存在下での抗腫瘍活性を測定した。また、比較
として本実施例と同一の健常人より得られた末梢血によ
り実施例４と同様の方法で得たＣＤ８陽性Ｔ細胞につい
ても上記３種類の樹立細胞への抗腫瘍活性も測定した。
測定は、γδ陽性Ｔ細胞についてはＥ／Ｔ比を２０：
１、１０：１及び５：１で反応させた。また、ＣＤ８陽
性Ｔ細胞については４０：１、２０：１及び１０：１で
反応させた。結果を表９に示す。OKT-3, K of cells obtained by the method
The antitumor activity of 562 and Daudi on 3 types of established cells was measured. Anti-C for K562 and Daudi
The antitumor activity in the presence of D3 antibody was measured. As a comparison, the antitumor activity of the above-mentioned three types of established cells was also measured for CD8-positive T cells obtained by the same method as in Example 4 using peripheral blood obtained from the same healthy person as in this example.
For the measurement, γδ-positive T cells had an E / T ratio of 20:
Reactions were carried out at 1, 10: 1 and 5: 1. Also, CD8-positive T cells were reacted at 40: 1, 20: 1 and 10: 1. The results are shown in Table 9.
【００５６】[0056]
【表９】 [Table 9]
【００５７】表９でγδ陽性Ｔ細胞とＣＤ８陽性Ｔ細胞
のＥ／Ｔ比２０：１及び１０：１の抗腫瘍活性値をみる
とγδ陽性Ｔ細胞のほうが高い抗腫瘍活性を示してい
る。更に、γδ陽性Ｔ細胞はＥ／Ｔ比を５：１にしても
抗腫瘍活性値は７５．３から９１．４％と、ＣＤ８陽性
Ｔ細胞のＥ／Ｔ比１０：１より高値を示している。When the antitumor activity values of γδ positive T cells and CD8 positive T cells at E / T ratios of 20: 1 and 10: 1 are shown in Table 9, the γδ positive T cells show higher antitumor activity. Furthermore, even if the E / T ratio of γδ-positive T cells was 5: 1, the antitumor activity value was 75.3 to 91.4%, which was higher than the E / T ratio of CD8-positive T cells of 10: 1. There is.
【００５８】本方法によりγδ陽性Ｔ細胞が純度９６．
８％で、しかも、吸着分離後に得られたリンパ球数の１
５００倍の３．０×１０9 個を得ることができた。更
に、該γδ陽性Ｔ細胞は、ＣＤ８陽性Ｔ細胞より抗腫瘍
活性が高いことがわかった。According to this method, γδ positive T cells have a purity of 96.
8% and 1 of the number of lymphocytes obtained after adsorption separation
It was possible to obtain 3.0 × 10 9 pieces of 500 times. Furthermore, it was found that the γδ positive T cells have higher antitumor activity than the CD8 positive T cells.
【００５９】[0059]
【発明の効果】以上説明した本発明によれば、目的の機
能的Ｔ細胞亜群が少量しか含まれないソ−スから該機能
的Ｔ細胞亜群を高純度にかつ大量に増殖培養することが
可能となった。当該方法で得られる高純度機能的Ｔ細胞
亜群は癌の養子免疫療法、Ｔ細胞抗原の機能解析の研究
等幅広い応用が期待される。EFFECTS OF THE INVENTION According to the present invention described above, it is possible to proliferate and culture a large amount of a target functional T cell subgroup from a source containing a small amount of the target functional T cell subgroup with high purity. Became possible. The highly purified functional T cell subgroup obtained by the method is expected to be widely applied in adoptive immunotherapy for cancer, research on functional analysis of T cell antigen, and the like.
Claims (6)
抗体固相化プレ−ト中で刺激させたのち増殖培養するこ
とを特徴とする機能的Ｔ細胞亜群の高純度大量培養方
法。1. Isolation of functional T cell subgroups for anti-CD3
A high-purity large-scale culture method for a functional T cell subgroup, which comprises stimulating in an antibody-immobilized plate and then proliferating and culturing.
％以上であることを特徴とする請求項１記載の方法で得
られる機能的Ｔ細胞亜群。2. The positive rate for functional T cell subgroups is 95.
% Or more, the functional T cell subgroup obtained by the method according to claim 1.
特徴とする請求項１記載の方法で得られる機能的Ｔ細胞
亜群。3. The functional T cell subgroup obtained by the method according to claim 1, which has a CD4 positive rate of 95% or more.
−フォリン陽性率が５０％以上であることを特徴とする
請求項１記載の方法で得られる機能的Ｔ細胞亜群。4. The functional T cell subgroup obtained by the method according to claim 1, wherein the CD4 positive rate is 95% or more and the perforin positive rate is 50% or more.
特徴とする請求項１記載の方法で得られる機能的Ｔ細胞
亜群。5. The functional T cell subgroup obtained by the method according to claim 1, wherein the CD8 positive rate is 95% or more.
徴とする請求項１記載の方法で得られる機能的Ｔ細胞亜
群。6. The functional T cell subgroup obtained by the method according to claim 1, which has a γδ positive rate of 95% or more.
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| AU746531B2 (en) * | 1998-03-12 | 2002-05-02 | Emory University | Methods and compositions for the selective expansion of gamma/delta T-cells |
| KR101299299B1 (en) * | 2007-11-05 | 2013-08-26 | 츠네오 쿠라모치 | The method for production of cell for cancer immunotherapy |
| JP5561845B2 (en) * | 2008-09-25 | 2014-07-30 | 株式会社リンフォテック | Formulation for increasing endogenous DHEA-S and method for producing the same, and method for increasing endogenous DHEA-S |
| JP6621171B2 (en) * | 2015-06-22 | 2019-12-18 | 国立大学法人広島大学 | T cell activator, T cell activation method, and T cell culture medium |
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1992
- 1992-02-05 JP JP4054433A patent/JP2530966B2/en not_active Expired - Lifetime
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| JPH05219945A (en) | 1993-08-31 |
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