JPWO2007060864A1 - Antibody-producing progenitor B cells with controllable proliferation and differentiation - Google Patents
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Abstract
本発明は、抗体医薬として利用可能なヒト型自己反応性抗体を作製する途を開く新規な抗体産生方法、およびその方法に有効に利用される抗体産生前駆細胞、並びに当該方法によって産生される抗体産生細胞を提供する。未分化Ｂ細胞の表面抗原マーカーを有する抗体産生前駆細胞をin vitroで作製し、これを免疫不全の非ヒト動物個体に移植して、当該動物の体内で抗体産生細胞に分化させることにより抗体を産生することができる。The present invention relates to a novel antibody production method that opens the way to produce a human-type autoreactive antibody that can be used as an antibody drug, an antibody-producing precursor cell that is effectively used in the method, and an antibody produced by the method. Producing cells are provided. An antibody-producing precursor cell having an undifferentiated B cell surface antigen marker is produced in vitro, transplanted into an immunodeficient non-human animal individual, and differentiated into an antibody-producing cell in the body of the animal. Can be produced.
Description
本発明は、新規な抗体産生技術に関する。より詳細には、本発明は、未分化Ｂ細胞の表面抗原マーカーを有する抗体産生前駆細胞をin vitroで作製し、これを動物個体に移植して生体内で抗体産生細胞に分化させる方法によって、抗体を産生する新規方法に関する。当該抗体産生技術は、抗体医薬として利用可能なヒト型自己反応性抗体を作製する途を開くものである。 The present invention relates to a novel antibody production technique. More specifically, the present invention relates to a method for producing an antibody-producing precursor cell having an undifferentiated B cell surface antigen marker in vitro, transplanting it to an animal individual, and differentiating it into an antibody-producing cell in vivo. It relates to a novel method for producing antibodies. This antibody production technique opens the way to produce human-type autoreactive antibodies that can be used as antibody drugs.
Ｂ細胞は、造血幹細胞からリンパ系幹細胞、プロＢ細胞、およびプレＢ細胞などを経て形質細胞へと分化し、抗体産生を行うことが知られている（非特許文献１参照）。形質細胞によって産生される抗体は各種解析に利用されるだけでなく、近年では抗体医薬や感染症等の診断薬として医療にも広く利用されており、抗体の需要は今後更に拡大することが予想される（非特許文献２参照）。抗体を、解析ではなく抗体医薬として利用しようとする際には、増殖因子受容体等の細胞表面のタンパク質を標的とする自己反応性抗体が必要とされることが多い（非特許文献３参照）。また、抗体医薬に利用する抗体は、免疫した宿主から得られる動物型の抗体ではなくヒト型である必要がある。これは、ヒト以外の型の抗体を投与すると、それ自身が抗原となり免疫応答を引き起こす危険性があるためである。 B cells are known to differentiate from hematopoietic stem cells to lymphoid stem cells, pro-B cells, pre-B cells, and the like into plasma cells to produce antibodies (see Non-Patent Document 1). Antibodies produced by plasma cells are not only used for various analyses, but in recent years they are also widely used in medicine as diagnostics for antibody drugs and infectious diseases, and the demand for antibodies is expected to expand further in the future. (See Non-Patent Document 2). When an antibody is to be used as an antibody drug rather than an analysis, an autoreactive antibody that targets a cell surface protein such as a growth factor receptor is often required (see Non-patent Document 3). . Moreover, the antibody used for the antibody drug needs to be a human type, not an animal type antibody obtained from an immunized host. This is because when a non-human type antibody is administered, it itself becomes an antigen and there is a risk of causing an immune response.
抗体の作製法としては、動物個体を免疫して形質細胞を単離し、不死化した細胞と細胞融合させてハイブリドーマを作製し、その中から目的の抗体を産生するクローンを選択する方法が一般的である。しかしながら、この従来の方法は以下に挙げる３つの問題を抱えている。 As a method for producing an antibody, a method is generally used in which an individual animal is immunized to isolate plasma cells, fused with immortalized cells to produce a hybridoma, and a clone that produces the target antibody is selected from the hybridoma. It is. However, this conventional method has the following three problems.
(1)医療に利用可能な自己反応性の機能抗体を得るのは種類によっては困難である、
(2)遺伝子組換えを行うことにより宿主動物型からヒト型の抗体へと変換する操作は確立されてはいるが（非特許文献４参照）、手間と時間を要する、
(3)医療への利用を目的とせず宿主動物型の抗体を得たい場合であっても、免疫過程、抗体産生細胞選択過程に時間がかかるため、目的の抗体を得るまでに長い日数を要する。(1) It is difficult to obtain a self-reactive functional antibody that can be used in medicine depending on the type.
(2) Although an operation for converting a host animal type into a human type antibody by genetic recombination has been established (see Non-Patent Document 4), it takes time and effort.
(3) Even if you want to obtain a host animal type antibody without aiming at medical use, it takes a long time to obtain the target antibody because the immunization process and the antibody-producing cell selection process take time. .
かかる問題点を解消するためには、動物個体を用いる従来の抗体作製方法では限界がある。
本発明は、in vitroでの新規な抗体産生方法を提供することを目的とする。好ましくは、抗体医薬として応用できる自己反応性抗体を作製可能な抗体産生方法、特にヒト型抗体の作製に応用可能な抗体産生方法を提供することを目的とする。また、本発明は抗体産生方法において得られる抗体産生細胞、ならびにその調製に使用される前駆細胞、好ましくは分化制御可能な前駆細胞を提供することを目的とする。 An object of the present invention is to provide a novel antibody production method in vitro. Preferably, an object of the present invention is to provide an antibody production method capable of producing an autoreactive antibody that can be applied as an antibody drug, particularly an antibody production method applicable to the production of a human-type antibody. Another object of the present invention is to provide antibody-producing cells obtained by the antibody production method, as well as progenitor cells used for the preparation thereof, preferably progenitor cells capable of controlling differentiation.
上記課題を解決するアプローチの一つとして、本発明者らは、未分化Ｂ細胞を不死化し、in vitroで分化誘導を行って抗体産生細胞へと分化させて抗体を得る方法を考えた。既に、鶏の未熟Ｂ細胞株であるDT40においては、ヒストン脱アセチル化酵素の阻害剤であるTrichostatin A（TSA）を用いた分化系が確立しているものの（Seo et al., 2005;参考文献１）、この方法は、特殊な鶏の細胞株を用いているため、ヒト形質細胞への応用が難しいという問題を含んでいる。 As one approach to solve the above problems, the present inventors have considered a method of obtaining an antibody by immortalizing undifferentiated B cells and inducing differentiation in vitro to differentiate into antibody-producing cells. DT40, an immature B cell line of chicken, has already established a differentiation system using Trichostatin A (TSA), an inhibitor of histone deacetylase (Seo et al., 2005; References) 1) This method has a problem that it is difficult to apply to human plasma cells because a special chicken cell line is used.
そこで本発明者らは、ヒト細胞への応用を考慮して、レトロウイルスベクターを用いて癌遺伝子を導入することによってマウス未分化Ｂ細胞を不死化する技術開発を試みたところ、特定のサイトカインの存在下でSV40ラージT抗原及びv-Ab1の遺伝子を導入することによって得られた不死化細胞がプロＢまたはプレＢの段階にあるＢ細胞の表現抗原マーカーを有しており、次いでこれを動物個体に移植したところ、当該動物の体内でIgMを発現するＢ細胞へと分化することを見出し、当該分化細胞が、抗体産生細胞として有用であること、また上記in vitroで作製されるＢ細胞の表現抗原マーカーを有する細胞が、当該抗体産生細胞を作製するための材料、特に自己産生するための材料として有用であることを確信した。また、本発明者らは、上記マウス未分化Ｂ細胞の不死化に用いる癌遺伝子として、温度変化もしくは薬剤投与による機能制御が可能な遺伝子を用いることによって、Ｂ細胞の増殖と分化が自由に制御できることを確認した。 Accordingly, the present inventors tried to develop a technique for immortalizing mouse undifferentiated B cells by introducing an oncogene using a retroviral vector in consideration of application to human cells. Immortalized cells obtained by introducing the gene of SV40 large T antigen and v-Ab1 in the presence have a B cell expression antigen marker at the pro-B or pre-B stage, When transplanted into an individual, it is found that the animal differentiates into a B cell that expresses IgM in the body of the animal, and that the differentiated cell is useful as an antibody-producing cell. It was convinced that the cells having the expression antigen marker were useful as a material for producing the antibody-producing cells, particularly as a material for self-production. In addition, the present inventors freely control the proliferation and differentiation of B cells by using a gene capable of controlling the function by temperature change or drug administration as an oncogene used for immortalization of the mouse undifferentiated B cells. I confirmed that I can do it.
本発明はかかる知見に基づいて完成したものであり、下記の具体的態様を有する。 The present invention has been completed based on such findings and has the following specific embodiments.
Ｉ．抗体産生前駆Ｂ細胞
（１）未分化Ｂ細胞を不死化することによって調製される、CD25またはB220のいずれかの表面抗原を発現してなる抗体非産生性の抗体産生前駆Ｂ細胞。
（２）SV40ラージT抗原および活性化型Ablが発現してなるプレＢ細胞である、（１）記載の抗体産生前駆Ｂ細胞。
（３）増殖が温度依存性であることを特徴とする、（１）または（２）記載の抗体産生前駆Ｂ細胞。
（４）SV40ラージT抗原が温度感受性である（２）または（３）記載の抗体産生前駆Ｂ細胞。
（５）抗体がＩｇＭである（１）乃至（４）のいずれかに記載の抗体産生前駆Ｂ細胞。
（６）マウス由来細胞である（１）乃至（５）のいずれかに記載する抗体産生前駆Ｂ細胞。
（７）被験動物に移植して当該動物の体内で分化させて抗体産生Ｂ細胞を作製するための（１）乃至（６）のいずれかに記載する抗体産生前駆Ｂ細胞。 I. Antibody-producing progenitor B cells (1) Non- antibody-producing antibody-producing progenitor B cells that are prepared by immortalizing undifferentiated B cells and that express either CD25 or B220 surface antigens.
(2) The antibody-producing precursor B cell according to (1), which is a pre-B cell obtained by expressing SV40 large T antigen and activated Abl.
(3) The antibody-producing precursor B cell according to (1) or (2), wherein proliferation is temperature-dependent.
(4) The antibody-producing precursor B cell according to (2) or (3), wherein the SV40 large T antigen is temperature sensitive.
(5) The antibody-producing precursor B cell according to any one of (1) to (4), wherein the antibody is IgM.
(6) The antibody-producing precursor B cell according to any one of (1) to (5), which is a mouse-derived cell.
(7) The antibody-producing precursor B cell according to any one of (1) to (6), which is transplanted into a test animal and differentiated in the body of the animal to produce an antibody-producing B cell.
II．抗体産生前駆Ｂ細胞の製造方法
（８）IL7の存在下で、SV40ラージT抗原の遺伝子と活性化型Ablの遺伝子を初代培養骨髄細胞に導入する工程を有する、CD25またはB220のいずれかの表面抗原を発現してなる抗体非産生性の抗体産生前駆Ｂ細胞を製造する方法。
（９）更に、上記工程で得られた初代培養骨髄細胞を培養する工程を有する、（８）に記載の方法。
（１０）SV40ラージT抗原が温度感受性である、（８）または（９）に記載する方法。
（１１）抗体がＩｇＭである（８）乃至（１０）のいずれかに記載する方法。
（１２）初代培養骨髄細胞がマウス由来細胞である（８）乃至（１１）のいずれかに記載する方法。 II. Method for Producing Antibody-Producing Progenitor B Cells (8) The surface of either CD25 or B220, which comprises the step of introducing SV40 large T antigen gene and activated Abl gene into primary cultured bone marrow cells in the presence of IL7 A method for producing a non-antibody-producing antibody-producing precursor B cell that expresses an antigen.
(9) The method according to (8), further comprising a step of culturing the primary cultured bone marrow cells obtained in the above step.
(10) The method according to (8) or (9), wherein the SV40 large T antigen is temperature sensitive.
(11) The method according to any one of (8) to (10), wherein the antibody is IgM.
(12) The method according to any one of (8) to (11), wherein the primary cultured bone marrow cells are mouse-derived cells.
III．抗体産生Ｂ細胞
（１３）（１）乃至（７）のいずれかに記載する抗体産生前駆Ｂ細胞を非ヒト免疫不全動物に移植して、当該動物の体内で分化させて製造される、抗体産生Ｂ細胞。 III. Antibody-producing B cells produced by transplanting antibody-producing precursor B cells described in any of (13) (1) to (7) into non-human immunodeficient animals and differentiating them in the body of the animals B cells.
IV．抗体産生Ｂ細胞の製造方法
（１４）（１）乃至（７）のいずれかに記載される抗体産生前駆Ｂ細胞を非ヒト免疫不全動物に移植して、当該動物の体内で抗体産生前駆細胞を抗体産生細胞に分化させることを特徴とする、抗体産生Ｂ細胞の製造方法。 IV. Method for producing antibody-producing B cells (14) The antibody-producing precursor B cells described in any one of (1) to (7) are transplanted into a non-human immunodeficient animal, and the antibody-producing precursor cells are produced in the body of the animal. A method for producing antibody-producing B cells, which comprises differentiating into antibody-producing cells.
V.抗体産生前駆細胞の使用
（１５）抗体産生B細胞の製造のための（１）乃至（７）のいずれかに記載される抗体産生前駆Ｂ細胞の使用。 V. Use of antibody-producing progenitor cells (15) Use of the antibody-producing progenitor B cells described in any of (1) to (7) for the production of antibody-producing B cells.
現在、抗体は、研究分野に限らず、感染症等の診断薬や抗体医薬などとして医療にも広く利用されており、抗体の需要は今後更に拡大することが予想される。前述するように、動物個体を免疫することに基づく従来の抗体作製方法は、抗体医薬等の医療に利用可能な自己反応性抗体の取得が難しいことに加えて、免疫過程や抗体産生細胞選択過程に時間がかかり、その取得には多大の時間と労力を要するという問題がある。 Currently, antibodies are not only used in research fields but also widely used in medicine as diagnostic agents for infectious diseases, antibody drugs, and the like, and the demand for antibodies is expected to further expand in the future. As described above, the conventional antibody production method based on immunizing an animal individual is difficult to obtain a self-reactive antibody that can be used for medical treatment such as an antibody drug, as well as an immunization process and an antibody-producing cell selection process. There is a problem that it takes a lot of time and much time and labor are required for the acquisition.
これに対して、本発明の抗体産生Ｂ細胞の製造方法ならびに抗体産生方法は、少ない労力で短期間に実施することができる。また本発明は、抗体産生細胞の作製に使用される前駆細胞をin vitroで調製し、免疫不全動物の体内で分化させて、抗体産生細胞ライブラリーを構築し、そのライブラリーの中から必要な抗体産生細胞を選択する方法であるため、自己反応性の機能抗体を調製することができる。特に本発明によれば、抗体産生Ｂ細胞としてＩｇＭ産生能を有するＢ細胞（IgM発現細胞）のライブラリーを提供することができる。 In contrast, the method for producing antibody-producing B cells and the method for producing antibodies of the present invention can be carried out in a short period of time with little effort. The present invention also provides a method for preparing precursor cells used for the production of antibody-producing cells in vitro and differentiating them in the body of an immunodeficient animal to construct an antibody-producing cell library. Since this is a method for selecting antibody-producing cells, a self-reactive functional antibody can be prepared. In particular, according to the present invention, a library of B cells (IgM-expressing cells) having IgM-producing ability can be provided as antibody-producing B cells.
本発明の抗体産生技術、並びに抗体産生細胞およびその前駆細胞は、医療分野で利用可能な抗体を作製する途を開くものとして意義あるものである。 The antibody production technique of the present invention, as well as antibody-producing cells and their precursor cells are significant as opening the way to produce antibodies that can be used in the medical field.
Ｉ．用語の説明
Abl
「Ab1」は、マウスで白血病を起こすエイベルソンマウス白血病ウイルスが発現するタンパク質である（Abelson and Rabstein, 1970；参考文献２；Goff et al., 1982；参考文献３）。このウイルスは、RNAウイルスであり、逆転写を受けた後、宿主のゲノム内に組み込まれる。ウイルスが発現するタンパク質と宿主（マウス）が発現するタンパク質とはそれをコードするゲノム配列が類似しており、ウイルスが保持する遺伝子を「v-abl」（そのタンパク質は「v-Abl」）、宿主が保持する遺伝子を「c-abl」（そのタンパク質は「c-Abl」）と称されている。また「v-Abl」は、チロシンリン酸化酵素であり、細胞の形質転換には、その酵素活性が必要であることがわかっている。 I. Explanation of terms
Abl
“Ab1” is a protein expressed by Abelson murine leukemia virus that causes leukemia in mice (Abelson and Rabstein, 1970;
v-Ablとc-Abl
「c-Abl」は、N末端からSH3ドメイン、SH2ドメイン、キナーゼドメイン、プロリンリッチ領域、DNA結合領域、およびアクチン結合領域を有するタンパク質である ( reviewed in Hantschel and Superti-furga, 2004；参考文献４) 。一方のv-Ablは、上記c-AblのSH3ドメインが欠損しており、代わりにウイルス由来のGagタンパク質が融合している構造を有する (Shore et al., 2002；参考文献５)。c-Ablの酵素活性は厳密に制御されているため、細胞に過剰発現させても細胞内のチロシンリン酸化レベルはほとんど変わらないが、一方のv-Ablは恒常的な酵素活性を保持するため、細胞に過剰発現させると細胞内のチロシンリン酸化レベルが上昇する。本発明において「活性化型Abl」とは、c-Ablのチロシンリン酸化活性がより活性化され、より高いチロシンリン酸化活性を有するものを意味し、v-Abl、Bcr-AblおよびTel-Ab1はその好適なものとして例示することができる。また、c-AblのSH3ドメインを欠損したり、SH3ドメインに点変異を導入して、SH3ドメインの機能を欠失した変異型は、v-Ablと同等の恒常的チロシンリン酸化活性を有することから（Jackson and Baltimore, 1989；参考文献６）、本発明でいう「活性型Abl」に含まれる。 v-Abl and c-Abl
“C-Abl” is a protein having an SH3 domain, SH2 domain, kinase domain, proline-rich region, DNA-binding region, and actin-binding region from the N-terminus (reviewed in Hantschel and Superti-furga, 2004; Reference 4 ) One v-Abl lacks the SH3 domain of the c-Abl and has a structure in which a virus-derived Gag protein is fused instead (Shore et al., 2002; Reference 5). Since c-Abl's enzymatic activity is strictly controlled, the level of tyrosine phosphorylation in the cell hardly changes even if it is overexpressed in the cell, but one v-Abl retains constitutive enzyme activity. When overexpressed in a cell, the level of tyrosine phosphorylation in the cell increases. In the present invention, the term “activated Abl” means that the tyrosine phosphorylation activity of c-Abl is more activated and has higher tyrosine phosphorylation activity, and v-Abl, Bcr-Abl and Tel-Ab1 Can be illustrated as a suitable example thereof. In addition, mutants lacking the SH3 domain function by deleting the SH3 domain of c-Abl or by introducing point mutations into the SH3 domain should have constant tyrosine phosphorylation activity equivalent to v-Abl. (Jackson and Baltimore, 1989; Reference 6), it is included in the “active Abl” in the present invention.
ところで「活性型Abl」の一つであるv-Ablは、in vivoまたin vitroにおいて、プレＢ細胞を癌化（不死化）し、その結果、成熟Ｂ細胞に分化するために必要な免疫グロブリン遺伝子の再構成が生じないことが知られている（Rosenberg, 1994;参考文献７）。AblのインヒビターであるSTI571(Imanitib, Gleevec)（Druker et al., 1996;参考文献８）で処理することによって、v-Ablで癌化したプレＢ細胞は、再び免疫グロブリンの再構成を始めることからも、v-AblがプレＢ細胞の癌化にとって必要であるといえる（Muljo and Schlissel, 2003；参考文献９）。 By the way, v-Abl, which is one of the “active Abl”, is an immunoglobulin necessary for in vivo or in vitro canceration (immortalization) of pre-B cells and, as a result, differentiation into mature B cells. It is known that gene rearrangement does not occur (Rosenberg, 1994; reference 7). By treating with STI571 (Imanitib, Gleevec), an inhibitor of Abl (Druker et al., 1996; Reference 8), pre-B cells that have become cancerous with v-Abl begin to reconstitute immunoglobulins again. Therefore, it can be said that v-Abl is necessary for canceration of pre-B cells (Muljo and Schlissel, 2003; Reference 9).
SV40
SV40は、ポリオワクチン作成の際に用いられたサルの腎臓細胞への汚染ウイルスとして見つかったウイルスである。ハムスターに感染させると腫瘍を形成することから、ワクチン接種を受けた人に対する癌化の観点で初期の研究が進行された。 SV40
SV40 is a virus found as a virus that contaminates monkey kidney cells used in the production of polio vaccines. Early studies were undertaken from the perspective of canceration in vaccinated people, as hamsters form tumors when infected.
SV40 T 抗原
試験管内での細胞レベルでの癌化を形質転換というが、上記SV40の形質転換能（癌化能）は主にT抗原にあることが知られている（Graham et al., 1975；参考文献１０）。SV40 T抗原遺伝子は、オルタナティブ・スプライシングによりLarge T抗原(LT)とSmall T抗原（ST）を発現する（Rundell and Parakati, 2001；参考文献１１）。LTは癌抑制遺伝子産物であるp53とRBに結合し不活性化することが知られている。また、STはセリン・スレオニンホスファターゼであるPP2A に結合し不活性化することが知られている。 SV40 T antigen Cancer transformation at the cell level is called transformation. However, the SV40 transformation ability (carcinogenicity) is known to be mainly in the T antigen (Graham et al., 1975). Reference 10). The SV40 T antigen gene expresses Large T antigen (LT) and Small T antigen (ST) by alternative splicing (Rundell and Parakati, 2001; Reference 11). LT is known to bind to and inactivate tumor suppressor gene products p53 and RB. ST is known to bind to and inactivate PP2A, a serine / threonine phosphatase.
初代培養細胞の不死化
初代培養細胞は、試験管内で数回継代を重ねると細胞老化を起こすことが知られている。マウス胎児繊維芽細胞（MEFs）は、p19ARF/p53経路が不活性化されると、老化を乗り越え不死化することが知られている（Kamijo et al., 1997；参考文献１２）。また、p53ノックアウトマウスから調製したMEFsは、細胞老化を経ることなく増殖する。 Immortalization of primary cultured cells It is known that primary cultured cells undergo cell senescence when they are passaged several times in a test tube. Mouse fetal fibroblasts (MEFs) are known to overcome senescence and become immortalized when the p19 ARF / p53 pathway is inactivated (Kamijo et al., 1997; reference 12). In addition, MEFs prepared from p53 knockout mice proliferate without undergoing cellular senescence.
なお、SV40 T抗原（主にLT）は、p53を不活性化することによって、げっ歯類の初代細胞を不死化させることが、変異型T抗原を用いた実験より明らかにされている(Jat and Sharp, 1986；参考文献１３)。 In addition, it has been clarified in experiments using mutant T antigen that SV40 T antigen (mainly LT) immortalizes primary cells of rodents by inactivating p53 (Jat and Sharp, 1986; reference 13).
温度感受性SV40 T抗原
SV40 T抗原の機能として初期に注目されたのは、ウイルスゲノム複製における役割である。温度感受性変異株を用いた実験から、許容温度下の33.5℃ではウイルスの複製が起こるが、非許容温度下の39℃では複製が起こらない変異株が見つかった。その温度感受性変異株の一つからクローニングされT抗原の変異が同定されたのが、tsA58(以下、tsTと省略)である（Tegtmeyer and Ozer, 1971;参考文献１４）。tsTは許容温度下の33.5℃では発現がみられるが、非許容温度下の39℃では発現が消失する。これは、tsTが39℃で分解しやすいためだと考えられている (Ray et al., 1992；参考文献１５)。 Temperature sensitive SV40 T antigen
The initial attention as a function of the SV40 T antigen is its role in viral genome replication. Experiments using temperature-sensitive mutants found mutants that replicated at 33.5 ° C under permissive temperature but did not replicate at 39 ° C under non-permissive temperature. It was tsA58 (hereinafter abbreviated as tsT) that was cloned from one of the temperature-sensitive mutants and identified as a T antigen mutation (Tegtmeyer and Ozer, 1971; Reference 14). The expression of tsT is observed at 33.5 ° C. under the permissible temperature, but the expression disappears at 39 ° C. under the non-permissive temperature. This is thought to be because tsT is easily decomposed at 39 ° C. (Ray et al., 1992; Reference 15).
tsTは、初期の研究においてはウイルスの機能を知るために用いられたが、その後、細胞の不死化などに利用されている。Jat等は、tsTを発現した初代細胞は、許容温度下の33.5℃では細胞が増殖するが、非許容温度下の39℃では増殖が停止することをみつけた（Jat and Sharp, 1989；参考文献１６）。このためtsTの利点として下記を挙げることができる。
(1)細胞の増殖を制御することができるため、例えば、目的細胞の増殖を停止させたり、分化誘導することが可能となる、
(2)Ｔ抗原を発現することで不死化した細胞について、不死化の原因が導入したＴ抗原の発現にあるか否かを検証することができる。Although tsT was used in early studies to know the function of the virus, it was later used to immortalize cells. Jat et al. Found that primary cells expressing tsT grew at 33.5 ° C under permissive temperature, but stopped at 39 ° C under non-permissive temperature (Jat and Sharp, 1989; References) 16). For this reason, the following can be mentioned as advantages of tsT.
(1) Since the growth of cells can be controlled, for example, it becomes possible to stop the growth of target cells or induce differentiation.
(2) For cells immortalized by expressing T antigen, it is possible to verify whether the cause of immortalization is the expression of the introduced T antigen.
前述のJat等は、この技術を個体に発展させ、主要組織適合遺伝子複合体H-2Kbのプロモーターの下流でtsTを発現するトランスジェニックマウスを作成し、ほぼ全ての組織でtsTを発現させることを可能にした。これにより様々な組織から得られた初代細胞は、tsTを発現しているため許容温度下の33.5℃で不死化していることが示された（Jat et al., 1991；参考文献１７）。またtsTトランスジェニックラットなども作成され、様々なセルラインが樹立されている（Obinata, 2001；参考文献１８）。Jat等は、tsTとヒトテロメレースの補酵素であるhTERTを用いることにより、不死化が難しいと言われているヒト細胞を不死化することにも成功している（O'Hare et al., 2001；参考文献１９)。The above-mentioned Jat et al. Develop this technology to individuals, create transgenic mice that express tsT downstream of the promoter of the major histocompatibility complex H-2K b , and express tsT in almost all tissues. Made possible. As a result, it was shown that primary cells obtained from various tissues were immortalized at 33.5 ° C. under permissive temperature because they expressed tsT (Jat et al., 1991; Reference 17). In addition, tsT transgenic rats and the like have been created, and various cell lines have been established (Obinata, 2001; Reference 18). Jat et al. Have succeeded in immortalizing human cells that are said to be difficult to immortalize by using hTERT, a coenzyme for tsT and human telomerase (O'Hare et al., 2001 Reference 19).
サイトカイン「IL-3」
「IL-3」は、Ｔ細胞において20αヒドロキシステロイド脱水素酵素（20α-SDH）を誘導する液性因子として同定された約25kDaの糖タンパク質であり、そのcDNAは1984年に単離された。IL-3の主な作用は血液細胞の増殖である。IL-3はその発現がT細胞や肥満細胞特異的で抗原刺激依存的であることから、炎症時などに働く反応性のサイトカインと考えられている。その作用は造血幹細胞を含む比較的未分化な造血細胞の増殖・分化の促進、肥満細胞の増殖促進、ヒスタミン放出の刺激などが知られている。また、IL-3のＢ細胞の増殖・分化に対する作用は、in vitroにおいて、マウスリンパ系前駆細胞のＢ細胞への分化を阻害するとの報告がある一方で、ヒトリンパ系前駆細胞においては分化を促進するという報告もあり、IL-3のＢ細胞に対する作用は未だ不明ではある。 Cytokine "IL-3"
“IL-3” is an approximately 25 kDa glycoprotein identified as a humoral factor that induces 20α hydroxysteroid dehydrogenase (20α-SDH) in T cells, and its cDNA was isolated in 1984. The main effect of IL-3 is blood cell proliferation. IL-3 is considered to be a reactive cytokine that acts during inflammation because its expression is specific to T cells and mast cells and is dependent on antigen stimulation. The action is known to promote the proliferation and differentiation of relatively undifferentiated hematopoietic cells including hematopoietic stem cells, promote the proliferation of mast cells, and stimulate histamine release. In addition, the effect of IL-3 on the proliferation and differentiation of B cells has been reported to inhibit the differentiation of mouse lymphoid progenitor cells into B cells in vitro, while human lymphoid progenitor cells promote differentiation. However, the effect of IL-3 on B cells is still unclear.
サイトカイン「IL-6」
「IL-6」は、Ｂ細胞の形質細胞への最終分化を誘導するＢ細胞分化因子（BSF-2）としてcDNAがクローニングされたサイトカインである。その後、IL-6は免疫応答のみならず造血系および神経系の細胞増殖や分化、急性期反応に関与し、さらに種々の免疫異常や炎症性疾患、リンパ系腫瘍の発症とも関係していることが明らかになった。IL-6はB細胞の増殖や分化に関わる因子として重要であるとされている。特に分化に対する作用としては、未成熟B細胞から成熟Ｂ細胞への分化に関与するとの報告がある。 Cytokine "IL-6"
“IL-6” is a cytokine in which cDNA is cloned as a B cell differentiation factor (BSF-2) that induces terminal differentiation of B cells into plasma cells. Later, IL-6 is involved not only in immune responses but also in hematopoietic and nervous system cell proliferation and differentiation, and acute phase reactions, and is also involved in the development of various immune abnormalities, inflammatory diseases, and lymphoid tumors. Became clear. IL-6 is considered to be important as a factor involved in B cell proliferation and differentiation. In particular, as an effect on differentiation, there is a report that it is involved in differentiation from immature B cells to mature B cells.
サイトカイン「IL-7」
「IL-7」は骨髄のストローマから分泌され、プレＢ細胞の増殖を促進するサイトカインとして、1988年にcDNAが単離された（Namen et al., 1988；参考文献２０）。IL-7のmRNAが胸腺にも発現していることからT細胞の発達との関わりについても調べられた結果、胸腺細胞や成熟T細胞に対し、増殖促進因子として作用することも報告された。ヒトIL-7は177アミノ酸として作られた後、Ｎ末端のシグナルペプチドが切断され、最終的に152アミノ酸が分泌される。ヒトとマウスのIL-7は、アミノ酸レベルでは60%の相同性がある。また、ヒトIL-7はヒトとマウスの両方で活性を示すが、マウスIL-7はヒトに対しては活性がないことが知られている。IL-7はＨ鎖再構成が始まるプロＢ細胞の時期に必須の因子であり（Grabstein et al., 1993; 参考文献２１）、細胞の増殖を促進することが知られている。IL-7を欠損したIL-7ノックアウトマウスは、極端なリンパ球減少症およびリンパ期間の形成不全を呈する（von Freeden-Jeffry et al., 1995；参考文献２２）。 Cytokine "IL-7"
"IL-7" is secreted from bone marrow stroma, and cDNA was isolated in 1988 as a cytokine that promotes proliferation of pre-B cells (Namen et al., 1988; Reference 20). Since IL-7 mRNA is also expressed in the thymus, its relationship with T cell development was also examined. As a result, it was reported that it acts as a growth promoting factor on thymocytes and mature T cells. Human IL-7 is produced as 177 amino acids, then the N-terminal signal peptide is cleaved, and finally 152 amino acids are secreted. Human and mouse IL-7 have 60% homology at the amino acid level. In addition, human IL-7 is active in both humans and mice, but mouse IL-7 is known to have no activity on humans. IL-7 is an essential factor during the pro-B cell period when heavy chain reorganization begins (Grabstein et al., 1993; Reference 21), and is known to promote cell proliferation. IL-7 knockout mice deficient in IL-7 display extreme lymphopenia and dysplasia of the lymph period (von Freeden-Jeffry et al., 1995; reference 22).
II．抗体産生前駆Ｂ細胞及びその製造方法
本発明が提供する抗体産生前駆Ｂ細胞は、それ自体に抗体を産生する能力はないものの、抗体を産生するＢ細胞（抗体産生Ｂ細胞）へと分化する能力を潜在的に有しているＢ細胞である。すなわち本発明の抗体産生前駆Ｂ細胞は、抗体産生Ｂ細胞の前駆細胞として位置づけることができる。なおここで抗体は、好適にはＩｇＭである。 II. Antibody producing precursor B cell and method for producing the same The antibody producing precursor B cell provided by the present invention does not have the ability to produce an antibody by itself, but can differentiate into an antibody producing B cell (antibody producing B cell). B cells that potentially have That is, the antibody-producing precursor B cell of the present invention can be positioned as a precursor cell of the antibody-producing B cell. Here, the antibody is preferably IgM.
具体的には、当該抗体産生前駆Ｂ細胞は、抗体（IgM）は発現しないものの、表面抗原として、Ｂ細胞でそれぞれの分化段階を通じて広く発現しＢ細胞のマーカーとして汎用されている表面抗原B220、またはプロＢ〜プレＢ細胞で発現する表面抗原CD25のいずれか少なくとも１つを発現していることを特徴とする。 Specifically, although the antibody-producing precursor B cell does not express an antibody (IgM), the surface antigen B220, which is widely expressed as a surface antigen in B cells through each differentiation stage and is widely used as a marker for B cells, Alternatively, at least one of the surface antigen CD25 expressed in pro-B to pre-B cells is expressed.
かかる性質を有する抗体産生前駆Ｂ細胞は、未分化Ｂ細胞を不死化することによって調製することができる。 Antibody-producing progenitor B cells having such properties can be prepared by immortalizing undifferentiated B cells.
未分化Ｂ細胞としては、動物、好ましくは哺乳動物の骨髄細胞から採取される造血幹細胞またはリンパ系前駆細胞を挙げることができる。好ましくは非ヒト哺乳動物に由来する造血幹細胞またはリンパ系前駆細胞である。より好ましくはラット、マウス、ハムスター、ウサギ、モルモット等のげっ歯動物、特に好ましくはマウスに由来する造血幹細胞またはリンパ系前駆細胞である。 Examples of undifferentiated B cells include hematopoietic stem cells or lymphoid progenitor cells collected from bone marrow cells of animals, preferably mammals. Hematopoietic stem cells or lymphoid progenitor cells derived from non-human mammals are preferred. More preferred are hematopoietic stem cells or lymphoid progenitor cells derived from rodents such as rats, mice, hamsters, rabbits and guinea pigs, particularly preferably mice.
未分化Ｂ細胞を不死化する方法は、特に制限されないが、好ましくは未分化Ｂ細胞を癌化する方法を挙げることができる。 The method for immortalizing undifferentiated B cells is not particularly limited, and a method for cancerating undifferentiated B cells is preferable.
未分化Ｂ細胞の癌化には、Ｂ細胞を癌化（不死化）する性質を有する活性化型Ablを用いることができる。具体的には、未分化Ｂ細胞に活性化型Ablの遺伝子を導入して発現させる方法を挙げることができる。かかる活性化型Ablとしては、マウスにおいてプレＢ細胞を癌化（不死化）することが知られているv-Abl（Rosenberg, 1994）、c-AblのSH3ドメインを欠損する等によってチロシンリン酸化活性がc-Ablに比して上昇してなる各種c-Abl変異型（Jackson and Baltimore, 1989；参考文献２３）、Bcr-Abl、およびTel-Ab1を挙げることができる。そして、ここでいうプレB細胞は、例えば、これに限定はされないが、表面抗原マーカがB200ポジティブ、CD25ポジティブ、IgMネガティブな細胞を含む細胞、として定義できる（Martensson, 2000；参考文献２４）。 For the transformation of undifferentiated B cells, activated Abl having the property of making B cells cancerous (immortalized) can be used. Specifically, a method of introducing and expressing an activated Abl gene into undifferentiated B cells can be mentioned. As such activated Abl, v-Abl (Rosenberg, 1994), which is known to cancerize (immortalize) pre-B cells in mice, tyrosine phosphorylation by deletion of the SH3 domain of c-Abl, etc. Examples include various c-Abl mutants (Jackson and Baltimore, 1989; Reference 23), Bcr-Abl, and Tel-Ab1 whose activity is increased compared to c-Abl. The pre-B cell here can be defined, for example, as a cell including, but not limited to, a cell whose surface antigen marker is B200 positive, CD25 positive, or IgM negative (Martensson, 2000; Reference 24).
好ましくは、v-Ablである。ここでBcr-Ablは、ヒトにおいて癌遺伝子として機能し（Wong and Witte, 2004;参考文献２５）、Ｂ細胞を癌化することが知られている活性化型Ablである。Tel-AblもBcr-Ablと同様に、ヒトにおいて癌遺伝子として機能し、リンパ細胞を癌化することが知られている活性化型Ablである。よって、未分化Ｂ細胞としてヒト由来の細胞を用いる場合、その癌化（不死化）にはBcr-AblまたはTel-Ablを好適に使用することができる。 Preferably, it is v-Abl. Here, Bcr-Abl is an activated Abl that functions as an oncogene in humans (Wong and Witte, 2004; Reference 25) and is known to cancerize B cells. Tel-Abl, like Bcr-Abl, is an activated Abl that functions as an oncogene in humans and is known to cancerize lymphocytes. Therefore, when human-derived cells are used as undifferentiated B cells, Bcr-Abl or Tel-Abl can be preferably used for their canceration (immortalization).
なお、未分化Ｂ細胞の不死化（癌化）は、人為的に制御できることが好ましい。未分化Ｂ細胞の不死化（癌化）を制御する方法としては、上記活性化型Ablの機能発現をAbl阻害剤によって制御する方法を挙げることができる。Abl阻害剤としては、制限されないが、例えばSTI571（Imatinib, Gleevec）(Druker et al., 1996;参考文献２６）、PD173955 (Nagar et al., 2002;参考文献２７)、AMN107 (Weisberg et al., 2005；参考文献２８)、BMS-354825(Shah et al., 2004; 参考文献２９)、ON012380(Gumireddy et al., 2005;参考文献３０)、PD166326(Wolff et al., 2005;参考文献３１)、およびAP23464 (O’Hare et al., 2004；参考文献３２)を例示することができる。 In addition, it is preferable that immortalization (canceration) of undifferentiated B cells can be artificially controlled. Examples of a method for controlling immortalization (canceration) of undifferentiated B cells include a method of controlling functional expression of the activated Abl with an Abl inhibitor. Examples of Abl inhibitors include, but are not limited to, STI571 (Imatinib, Gleevec) (Druker et al., 1996; Reference 26), PD173955 (Nagar et al., 2002; Reference 27), AMN107 (Weisberg et al. 2005; Reference 28), BMS-354825 (Shah et al., 2004; Reference 29), ON012380 (Gumireddy et al., 2005; Reference 30), PD166326 (Wolff et al., 2005; Reference 31) ), And AP23464 (O'Hare et al., 2004; reference 32).
ところで、活性化型Abl、特にv-Ablを未分化Ｂ細胞（リンパ系前駆細胞）に感染させると、癌遺伝子に対する防御機構として、p53依存的なアポトーシスが生じる（Radfar et al., 1998；参考文献３３）。このため、未分化Ｂ細胞の不死化（癌化）に際しては、未分化Ｂ細胞への上記活性化型Abl遺伝子の導入と並行して、p53を不活性化することでアポトーシスを防止することが好ましい。 By the way, when activated Abl, especially v-Abl, is infected to undifferentiated B cells (lymphoid precursor cells), p53-dependent apoptosis occurs as a defense mechanism against oncogenes (Radfar et al., 1998; reference) Reference 33). Therefore, upon immortalization (canceration) of undifferentiated B cells, it is possible to prevent apoptosis by inactivating p53 in parallel with the introduction of the activated Abl gene into undifferentiated B cells. preferable.
p53の不活性化処理は特に制限されないが、公知の方法を用いることが出来る。非ヒト細胞におけるp53の不活性化に関しては、遺伝子改変動物が利用可能である。具体的には、p53ノックアウトマウスやp53を不活性化する癌遺伝子を発現するトランスジェニックマウスやラットを挙げることが出来る。特にヒト細胞への応用も考慮するならば、優先不全型p53を発現させる、siRNAによりp53 mRNAのノックダウンを行うなどの方法を挙げることが出来る。また、p53のシグナル伝達経路に関わる分子の制御に関しては、限定はされないが、p53の上流に位置しp53のユビキチンを介した分解に関与するMDM2を過剰発現する方法や、MDM2のネガティブレギュレーターであるp19をsiRNAにより不活性化するなどの方法を挙げることができる。ウイルス癌遺伝子を用いるp53の不活性化方法としては、限定はされないが、ヒトパピローマウイルスのE6を発現させる、アデノウイルスのE1Bを発現させるなどの方法をあげることが出来る。特に好ましくは、p53を不活性化することが知られているSV40 T抗原の遺伝子を初代骨髄細胞に導入して発現させることが出来る。T抗原は、オルタナティブ・スプライシングによりLarge T抗原(LT)とSmall T抗原(ST)を発現することが知られている。p53の不活性化にはLTが必要なことから、T抗原の中でもLTを好適に用いることが出来る。この時、T抗原の発現を自由に制御できることが好ましい。T抗原の発現制御に関しては、限定はされないが、温度制御により機能のON/OFFが可能な温度感受性Ｔ抗原(tsT)を用いることが出来る(Jat and Sharp, 1989；参考文献３４)。温度感受性変異部位はSTではなくLTに含まれるため、より好ましくは温度感受性のLT(tsLT)である。tsTは、ヒトへの応用も可能であり(O’Hare et al., 2001；参考文献３５)、不死化にはLTが大きく寄与するため、 本発明の技術(tsLT)は、ヒト細胞に応用可能であると考えられる。なおtsLTは、p53以外にもがん抑制遺伝子産物であるRBなどを不活性化することが知られており、tsLTを用いた場合には、p53不活性化以外の効果も期待することができる。 The inactivation treatment of p53 is not particularly limited, but a known method can be used. For inactivation of p53 in non-human cells, genetically modified animals are available. Specific examples include p53 knockout mice, transgenic mice and rats that express an oncogene that inactivates p53. In particular, if application to human cells is also considered, methods such as expressing preferentially deficient p53 and knocking down p53 mRNA with siRNA can be mentioned. In addition, the regulation of molecules involved in the signal transduction pathway of p53 is not limited, but it is a method of overexpressing MDM2 that is located upstream of p53 and involved in degradation of p53 via ubiquitin, and is a negative regulator of MDM2. Examples thereof include a method of inactivating p19 with siRNA. Methods for inactivating p53 using a viral oncogene include, but are not limited to, methods such as expressing human papillomavirus E6 and adenovirus E1B. Particularly preferably, the SV40 T antigen gene known to inactivate p53 can be introduced and expressed in primary bone marrow cells. T antigen is known to express Large T antigen (LT) and Small T antigen (ST) by alternative splicing. Since LT is required for inactivation of p53, LT can be preferably used among T antigens. At this time, it is preferable that the expression of T antigen can be freely controlled. The T antigen expression control is not limited, but a temperature sensitive T antigen (tsT) that can be turned ON / OFF by temperature control can be used (Jat and Sharp, 1989; Reference 34). Since the temperature-sensitive mutation site is contained in LT instead of ST, temperature-sensitive LT (tsLT) is more preferable. Since tsT can be applied to humans (O'Hare et al., 2001; Reference 35), and LT greatly contributes to immortalization, the technique of the present invention (tsLT) can be applied to human cells. It is considered possible. In addition to p53, tsLT is known to inactivate RB, which is a tumor suppressor gene product. When tsLT is used, effects other than p53 inactivation can be expected. .
未分化Ｂ細胞の不死化方法としては、具体的には、例えば上記活性化型AblとT抗原の両方の遺伝子を未分化Ｂ細胞を含む初代培養骨髄細胞に導入し、これらを発現させる方法等を挙げることができる。 As a method for immortalizing undifferentiated B cells, specifically, for example, a method of introducing both the activated Abl and T antigen genes into primary cultured bone marrow cells containing undifferentiated B cells and expressing them, etc. Can be mentioned.
未分化Ｂ細胞を含む初代培養骨髄細胞は、定法に従って調製することができる（例えば「機能細胞の分離と培養」、三井洋司ら、第131-138頁、1988年、丸善など参照）。 Primary cultured bone marrow cells containing undifferentiated B cells can be prepared according to a conventional method (see, for example, “Separation and culture of functional cells”, Yoji Mitsui et al., Pages 131-138, 1988, Maruzen, etc.).
また未分化Ｂ細胞を含む初代培養骨髄細胞は商業的に入手可能であり、簡便にはこれを用いることもできるし、また生体から直接取りだした骨髄細胞を市販の初代培養細胞培養キットを用いて調製することもできる。 In addition, primary cultured bone marrow cells containing undifferentiated B cells are commercially available, and can be used conveniently, or bone marrow cells taken directly from a living body can be obtained using a commercially available primary culture cell culture kit. It can also be prepared.
初代培養骨髄細胞（未分化Ｂ細胞）に活性化型AblとT抗原の遺伝子を導入する方法としては、特に制限されないが、好適にはレトロウイルスベクターを用いる方法を挙げることができる。具体的には、活性化型Ablの遺伝子とT抗原の遺伝子を有するレトロウイルスベクター、または活性化型Ablの遺伝子を有するレトロウイルスベクターとT抗原の遺伝子を有するレトロウイルスベクターとを調製し、これを用いて、初代培養骨髄細胞（未分化Ｂ細胞）を形質転換（感染）する方法を用いることができる。 A method for introducing activated Abl and T antigen genes into primary cultured bone marrow cells (undifferentiated B cells) is not particularly limited, and a method using a retroviral vector is preferable. Specifically, a retroviral vector having an activated Abl gene and a T antigen gene, or a retroviral vector having an activated Abl gene and a retroviral vector having a T antigen gene are prepared. Can be used to transform (infect) primary cultured bone marrow cells (undifferentiated B cells).
ここで活性化型Ablの遺伝子およびT抗原の遺伝子を導入するために用いられるレトロウイルスベクターとしては、制限されないが、一例として、pFB neoベクター Neomycin耐性(Stratagene)、pLNCX2ベクター Neomycin耐性(Clontech)、pLHCX ベクター HygromycinB耐性(Clontech)、pLPCX ベクター Puromycin耐性(Clontech)、pDON-AIベクター Neomycin耐性(Takara)、pBabe ベクター(Morgenstern and Land, 1990；参考文献３６)、pMX ベクター (Onishi et al., 1996；参考文献３７)、pCX ベクター(Akagi et al., 2000；参考文献３８)、pCX4 ベクター(Akagi et al., 2003；参考文献３９)、MSCVベクター(Hawley et al., 1994；参考文献４０)、MNDベクター(Halene et al., 1999；参考文献４１)、およびMFG-Sベクター(Riviere et al., 1995；参考文献４２)を挙げることができる。 Here, the retrovirus vector used to introduce the activated Abl gene and the T antigen gene is not limited.For example, pFB neo vector Neomycin resistance (Stratagene), pLNCX2 vector Neomycin resistance (Clontech), pLHCX vector Hygromycin B resistance (Clontech), pLPCX vector Puromycin resistance (Clontech), pDON-AI vector Neomycin resistance (Takara), pBabe vector (Morgenstern and Land, 1990; reference 36), pMX vector (Onishi et al., 1996; Reference 37), pCX vector (Akagi et al., 2000; reference 38), pCX4 vector (Akagi et al., 2003; reference 39), MSCV vector (Hawley et al., 1994; reference 40), Mention may be made of MND vectors (Halene et al., 1999; reference 41) and MFG-S vectors (Riviere et al., 1995; reference 42).
ここでレトロウイルスは増殖する細胞にしか目的の遺伝子を組み込むことができない。そこで初代培養骨髄細胞（未分化Ｂ細胞）を選択的に増殖させる方法として、初代培養骨髄細胞（未分化Ｂ細胞）をサイトカインで処理する方法を挙げることができる。かかる方法として、具体的には、初代培養骨髄細胞（未分化Ｂ細胞）をインフェクション（レトロウイルスベクターによる活性化型AblとT抗原の遺伝子の導入）の前に予めサイトカインで処理して増殖能を付与するか、またはサイトカイン存在下でインフェクション（レトロウイルスベクターによる活性化型AblとT抗原の遺伝子の導入）を行う方法を挙げることができる（例えば、「新細胞工学実験プロトコール」、東京大学医科学研究所 制癌研究部、第163-170頁、1993年、秀潤社）。 Here, a retrovirus can only integrate a target gene into a proliferating cell. Thus, as a method for selectively proliferating primary cultured bone marrow cells (undifferentiated B cells), a method of treating primary cultured bone marrow cells (undifferentiated B cells) with a cytokine can be mentioned. As such a method, specifically, primary cultured bone marrow cells (undifferentiated B cells) are treated with cytokines in advance before infection (introduction of activated Abl and T antigen genes by retroviral vectors) to increase proliferation ability. Or a method of infecting in the presence of cytokines (introduction of activated Abl and T antigen genes with a retroviral vector) (for example, “New Cell Engineering Experimental Protocol”, University of Tokyo Medical Science) Institute of Cancer Research, pp. 163-170, 1993, Shujunsha).
なお、ここで用いられるサイトカインとしては、Ｂ細胞の分化の初期に必要とされるInterleukin-7 （IL-7）、Flt3 ligand（FL）、kit ligand （SCF）を挙げることができる。好ましくはInterleukin-7（IL-7）である。 Examples of cytokines used here include Interleukin-7 (IL-7), Flt3 ligand (FL), and kit ligand (SCF) that are required in the early stages of B cell differentiation. Interleukin-7 (IL-7) is preferable.
斯くして調製される細胞の中から、上記サイトカイン、好ましくはInterleukin-7の存在下で浮遊細胞として増殖する細胞を選択することによって、CD25またはB220のいずれかの表面抗原を発現してなる本発明の抗体産生前駆Ｂ細胞として取得することができる。 A cell that expresses the surface antigen of either CD25 or B220 by selecting a cell that proliferates as a floating cell in the presence of the above cytokine, preferably Interleukin-7, from the cells thus prepared. It can be obtained as an antibody-producing precursor B cell of the invention.
当該抗体産生前駆Ｂ細胞は、それ自体に抗体を産生する能力はないものの、抗体を産生するＢ細胞（抗体産生Ｂ細胞）へと分化する能力を潜在的に有している。このため、抗体産生Ｂ細胞を作製するための材料として有効に利用することができる。 Although the antibody-producing precursor B cell itself does not have the ability to produce antibodies, it has the potential to differentiate into B cells that produce antibodies (antibody producing B cells). Therefore, it can be effectively used as a material for producing antibody-producing B cells.
III．抗体産生Ｂ細胞およびその製造方法
さらに本発明は、抗体産生Ｂ細胞およびその製造方法を提供する。本発明が提供する抗体産生Ｂ細胞は、抗体産生能、好適にはＩｇＭを産生する能力を有するものである。かかる抗体産生Ｂ細胞は、前記本発明の抗体産生前駆Ｂ細胞を免疫不全動物に移植して、当該動物の体内で分化させることによって製造することができる。 III. Antibody-Producing B Cell and Method for Producing the Same Further, the present invention provides an antibody-producing B cell and a method for producing the same. The antibody-producing B cell provided by the present invention has an antibody producing ability, preferably an ability to produce IgM. Such antibody-producing B cells can be produced by transplanting the antibody-producing precursor B cells of the present invention into an immunodeficient animal and differentiating them in the animal body.
ここで免疫不全動物としては、非ヒト動物、好ましくは非ヒト哺乳動物を挙げることができる。より好ましくはラット、マウス、ハムスター、ウサギ、モルモット等のげっ歯動物であり、特に好ましくはマウスである。なお、免疫不全マウスとして商業的に入手できるものとしてはNOD/SCIDマウス、SCIDマウス、およびnudeマウス（以上、日本クレアなど）を挙げることができる。 Here, examples of the immunodeficient animal include non-human animals, preferably non-human mammals. More preferred are rodents such as rats, mice, hamsters, rabbits and guinea pigs, and particularly preferred are mice. Examples of commercially available immunodeficient mice include NOD / SCID mice, SCID mice, and nude mice (Japan Clare, etc.).
抗体産生前駆Ｂ細胞の移植方法としては、特に制限されないが、例えば簡便な方法として、抗体産生前駆Ｂ細胞を静脈中に入れる方法や当該細胞の腹腔内注射などを挙げることができる。なお、免疫不全動物の飼育は特に制限されず、定法に従うことができる。例えば、感染予防を施した環境下で滅菌処理した飼料や水を摂取させて飼育する方法を挙げることができる。 The method for transplanting antibody-producing precursor B cells is not particularly limited, and examples of convenient methods include a method of putting antibody-producing precursor B cells into a vein and intraperitoneal injection of the cells. The breeding of immunodeficient animals is not particularly limited, and can follow a standard method. For example, a method of raising and sterilizing feed and water sterilized in an environment in which infection is prevented can be mentioned.
抗体産生Ｂ細胞は、免疫不全動物に抗体産生前駆Ｂ細胞を移植し、一定期間飼育して生体内で分化させた後、骨髄または脾臓から採取した骨髄細胞または脾臓細胞から調製することができる。なお、当該骨髄細胞または脾臓細胞は、制限されないが、通常、免疫不全動物に抗体産生前駆Ｂ細胞を移植した後、２週間程度の飼育後に回収することができる。骨髄細胞または脾臓細胞から抗体産生Ｂ細胞を調製する方法としては、特に制限されないが、抗ＩｇＭ抗体を指標として、FACS vantage (BD biosciences)で細胞を分取し、さらに必要に応じて分取した細胞をin vitroで増殖させる方法を例示することができる。 Antibody-producing B cells can be prepared from bone marrow cells or spleen cells collected from bone marrow or spleen after transplanting antibody-producing precursor B cells to immunodeficient animals, rearing them for a certain period of time and differentiating them in vivo. The bone marrow cells or spleen cells are not limited, but can usually be collected after breeding for about 2 weeks after transplanting antibody-producing precursor B cells into an immunodeficient animal. The method for preparing antibody-producing B cells from bone marrow cells or spleen cells is not particularly limited, but the cells were fractionated by FACS vantage (BD biosciences) using anti-IgM antibody as an index, and further fractionated as necessary. A method for growing cells in vitro can be exemplified.
斯くして調製される抗体産生Ｂ細胞は、抗体産生能、とくにＩｇＭを産生する能力を有しており、抗体産生材料として好適に使用することができる。特に当該抗体産生Ｂ細胞は、自己反応性抗体が産生できる可能性がある点で有用な抗体産生材料である。本発明が提供する抗体産生Ｂ細胞は、好適にはＩｇＭ発現細胞であるが、クラススイッチ誘導を行うことにより、ＩｇＧ発現細胞に変換することも可能である。なお、ＩｇＭ発現細胞からＩｇＧ発現細胞に変換するためのクラススイッチ誘導法については一部、効率は低いが論文で報告されている（Cerutti，et al.,1998；参考文献４３）。 The antibody-producing B cells thus prepared have an antibody-producing ability, particularly an ability to produce IgM, and can be suitably used as an antibody-producing material. In particular, the antibody-producing B cells are useful antibody-producing materials in that autoreactive antibodies can be produced. The antibody-producing B cells provided by the present invention are preferably IgM-expressing cells, but can be converted into IgG-expressing cells by inducing class switching. In addition, the class switch induction method for converting from an IgM-expressing cell to an IgG-expressing cell has been reported in a paper (Cerutti, et al., 1998; Reference 43), although the efficiency is partially low.
本発明の未分化B細胞を非ヒト免疫不全動物内で抗体産生細胞へと分化させる方法は、将来的に本来の動物個体の抗体産生システムでなく、インビトロで抗体産生細胞のライブラリーを作成する方法を提供し得る。また、本発明はヒト細胞にも適用し得る。ヒト未分化B細胞を非ヒト免疫不全動物内で抗体産生細胞へと分化させる方法はいまだ確立していない。ヒト細胞を使った該方法が実施できれば、ヒト抗体産生細胞を製造することも可能である。該ヒト抗体産生細胞が多様な抗体を産生する場合、抗体産生細胞はライブラリーとして製造し得る。そのライブラリーは抗体医薬のスクリーニングにおいて、基本的な抗体医薬の候補を提供しうる。従って、本発明の方法は、新規で画期的な抗体医薬の製造方法を提供するという産業上の利用可能性がある。 The method of differentiating undifferentiated B cells of the present invention into antibody-producing cells in a non-human immunodeficient animal creates a library of antibody-producing cells in vitro instead of the original antibody-producing system of individual animals in the future. A method may be provided. The present invention can also be applied to human cells. A method for differentiating human undifferentiated B cells into antibody-producing cells in non-human immunodeficient animals has not yet been established. If the method using human cells can be carried out, human antibody-producing cells can be produced. When the human antibody-producing cells produce various antibodies, the antibody-producing cells can be produced as a library. The library can provide basic antibody drug candidates in antibody drug screening. Therefore, the method of the present invention has industrial applicability to provide a novel and innovative method for producing an antibody drug.
以下、本発明をより詳細に示すために実験例を示す。 Hereinafter, experimental examples are shown in order to show the present invention in more detail.
なお、各操作は、下記の実験例に具体的に示す操作に限られず、遺伝子組換え技術の一般方法、例えば Molecular Cloning.(1989),(Cold Spring Harbor Lab.) に記載の方法に従って行うこともできる。これらの操作に使用されるベクター、発現キットおよびパッケージング細胞は商業的に入手可能であり、簡便にはそれを用いて製造販売者の推奨するマニュアルに従って実施することができる。例えば、クロンテック（株）からは、発現キットとして、パントロピックウィルス発現系、MSCVレトロウィルス発現系、RevTet-On & RevTet-Off遺伝子発現系が、またパッケージング細胞（システム）としてEcoPack2-293 Cell Line、AmphoPack-293 Cell Line、RetroPack PT67 Cell Line、Retro-X Universal Packaging Systemを入手することができる。またタカラバイオからはRetrovirus Packaging Kit Eco、Retrovirus Packaging Kit Amphoを、さらにINGENEXからはRetroMax（商標）Retrovirus Systemを入手することができる。 In addition, each operation is not limited to the operation specifically shown in the following experimental examples, but should be performed according to a general method of gene recombination technology, for example, the method described in Molecular Cloning. (1989), (Cold Spring Harbor Lab.). You can also. Vectors, expression kits and packaging cells used for these operations are commercially available, and can be conveniently used according to the manual recommended by the manufacturer. For example, from Clontech, pantropic virus expression system, MSCV retrovirus expression system, RevTet-On & RevTet-Off gene expression system as expression kits, and EcoPack2-293 Cell Line as packaging cells (system) AmphoPack-293 Cell Line, RetroPack PT67 Cell Line, and Retro-X Universal Packaging System are available. Moreover, Retrovirus Packaging Kit Eco and Retrovirus Packaging Kit Ampho can be obtained from Takara Bio, and RetroMax (trademark) Retrovirus System can be obtained from INGENEX.
なお、下記の実験例の記載において、「％」とは特に言及しない限り「重量％」を意味する。 In the description of the experimental examples below, “%” means “% by weight” unless otherwise specified.
実験例１ 不死化の検討
（１）骨髄細胞の調製
野生型マウス（C57BL/6J、日本クレア）から骨髄細胞を採取し、0.8% NH4Cl溶液を用いて赤血球画分を除去した。得られた白血球画分を、10 容量% FBS、50mM 2-Mercaptoethanol、および抗生物質 (Antibiotic-Antimycotic; Gibco）を添加したRPMI 1640（日研生物医学研究所）に懸濁し、これにIL-3、IL-6、IL-7を単独あるいは3種組み合わせて、最終濃度が培養液の2容量%になるようにコンディションド培地（下記参照）に添加し、33.5℃、5容量％CO2で培養した。なお、IL-3、IL-6、およびIL-7は、当該サイトカインをその培養上清に放出するサイトカイン過剰発現細胞（それぞれWI3、WI6、WI7: 東京医科歯科大の烏山先生より分与）を使って調製した。そして、当該サイトカインを含む培養上清（コンディションド培地）を骨髄細胞と共に培養した。 Experimental Example 1 Examination of Immortalization (1) Preparation of Bone Marrow Cells Bone marrow cells were collected from wild type mice (C57BL / 6J, Nippon Claire) and the erythrocyte fraction was removed using a 0.8% NH 4 Cl solution. The obtained leukocyte fraction was suspended in RPMI 1640 (Nikken Biomedical Research Institute) supplemented with 10 vol% FBS, 50 mM 2-Mercaptoethanol, and antibiotics (Antibiotic-Antimycotic; Gibco). , IL-6, IL-7, alone or in combination, add to conditioned medium (see below) so that the final concentration is 2% by volume of the culture, and culture at 33.5 ° C and 5% CO 2 did. IL-3, IL-6, and IL-7 are cytokine-overexpressing cells that release the cytokine into the culture supernatant (WI3, WI6, and WI7, respectively, distributed by Prof. Hiyama at Tokyo Medical and Dental University). Prepared. And the culture supernatant (conditioned medium) containing the said cytokine was cultured with the bone marrow cell.
（２）癌遺伝子tsLT発現ベクターの調製
癌遺伝子として導入したtsLTは、以下の方法で作成した。pZip neo SV40 tsT(University College London、Jat博士から分与)を鋳型に、Pfu ポリメラーゼ (Stratagene)を用いて１番目のエキソンを5’-ggatccagatggataaagttttaaacagagag-3’ （配列番号１）と 5’-ccataggttggaatctcagttgcatcccagaagcct-3’（配列番号２）のプライマー、２番目のエキソンを5’-ggatgcaactgagattccaacctatggaactgatgaat-3’（配列番号３）と5’-ggatccttatgtttcaggttcaggttcagggggag-3’（配列番号４）のプライマーで増幅後、それぞれの増幅された断片を更に5’-ggatccagatggataaagttttaaacagagag-3’（配列番号５） と 5’-ggatccttatgtttcaggttcaggttcagggggag-3’（配列番号６）のプライマーで増幅し、得られた完全長のtsLTをpCRBlunt (Invitrogen) に挿入し、そのシークエンスを確認した。その後、得られた完全長のtsLTをレトロウイルスベクターpCX4 puro（大阪バイオサイエンス研究所、赤城先生より分与）に挿入した（以下、このtsLT発現べクターを、「pCX4puro tsLT」という）。このべクターを発現する細胞は、puromycinに対する抵抗性を持つので、薬剤選択が可能である。(2) Preparation of Oncogene tsLT Expression Vector tsLT introduced as an oncogene was prepared by the following method. Using pZip neo SV40 tsT (university from University College London, Dr. Jat) as a template and using Pfu polymerase (Stratagene), the first exon is 5'-ggatccagatggataaagttttaaacagagag-3 '(SEQ ID NO: 1) and 5'-ccataggttggaatctcagttgcatcccagaagcct- After amplification with the primer of 3 '(SEQ ID NO: 2) and the second exon with the primers of 5'-ggatgcaactgagattccaacctatggaactgatgaat-3' (SEQ ID NO: 3) and 5'-ggatccttatgtttcaggttcaggttcagggggag-3 '(SEQ ID NO: 4) The fragment was further amplified with primers 5'-ggatccagatggataaagttttaaacagagag-3 '(SEQ ID NO: 5) and 5'-ggatccttatgtttcaggttcaggttcagggggag-3' (SEQ ID NO: 6), and the resulting full-length tsLT was inserted into pCRBlunt (Invitrogen). , Confirmed the sequence. Thereafter, the obtained full-length tsLT was inserted into a retrovirus vector pCX4 puro (distributed by Osaka Bioscience Institute, Prof. Akagi) (hereinafter, this tsLT expression vector is referred to as “pCX4puro tsLT”). Cells that express this vector have resistance to puromycin, so drug selection is possible.
（３）v-Abl発現ベクターの調製
v-Abl（Columbia University、Goff博士より分与）を、レトロウイルスベクターpCX GFPに挿入することによりv-Abl発現ベクターを作成した（以下、このv-Abl発現ベクターを、「pCX GFP v-Abl」という）。なお、レトロウイルスベクターpCX GFPは、pEGFP-C1 (BDBiosciences) から切り出したEGFP断片を、bsr領域を取り除いたpCX bsrベクター（大阪バイオサイエンス研究所、赤城先生より分与）に挿入することで作成した。このべクター（pCX GFP v-Abl）を発現する細胞は、GFP（Green Fluorescent Protein）を発現するので、セルソーターにより分取が可能である。(3) Preparation of v-Abl expression vector
A v-Abl expression vector was constructed by inserting v-Abl (distributed by Columbia University, Dr. Goff) into the retroviral vector pCX GFP (hereinafter referred to as “pCX GFP v-Abl”). "). The retroviral vector pCX GFP was created by inserting the EGFP fragment excised from pEGFP-C1 (BDBiosciences) into the pCX bsr vector (distributed by Prof. Akagi, Osaka Bioscience Laboratories) with the bsr region removed. . Cells expressing this vector (pCX GFP v-Abl) express GFP (Green Fluorescent Protein) and can be sorted using a cell sorter.
（４）インフェクション
ウイルス感染のため、tsLT発現べクター（pCX4 puro tsLT）をpGPおよびpeEco（Takara）とともにFugene6 (Roche)を用いてレトロウィルスパッケージング細胞、PLT-E（東京大学医学研究所、北村先生より分与）にトランスフェクションした。具体的には、pCX4 puro tsLT、pGP、peEco（Takara）、Fugene6 (Roche)およびPLT-Eを培養液（10% FBSと抗生物質[Antibiotic-Antimycotic; Gibco]を含むDMEM）に懸濁して、37℃、5容量％CO2で培養し、24時間後、培養液を交換し、更に24時間培養した後、培養上清を回収した。得られたウイルスを含む培養上清を、4℃, 8000 gで16 時間遠心し濃縮させて、細胞に感染させた。(4) Infection For virus infection, retrovirus packaging cells, PLT-E (University of Tokyo, Kitamura) using Fugene6 (Roche) with pGP and peEco (Takara) for tsLT expression vector (pCX4 puro tsLT) Transfection was performed by the teacher. Specifically, pCX4 puro tsLT, pGP, peEco (Takara), Fugene6 (Roche) and PLT-E are suspended in a culture solution (DMEM containing 10% FBS and antibiotics [Antibiotic-Antimycotic; Gibco]), After culturing at 37 ° C. and 5% by volume of CO 2 , 24 hours later, the culture solution was changed, and further cultured for 24 hours, and then the culture supernatant was collected. The obtained culture supernatant containing the virus was centrifuged at 4 ° C. and 8000 g for 16 hours and concentrated to infect cells.
また、pCX4 puro tsLTウイルス感染2日後、この細胞にv-Abl発現ベクター（pCX v-Abl GFP）を、上記と同様の方法でPLT-E細胞にインフェクションして得られたウイルスを含む培養上清を濃縮後、細胞に感染させた。 Also, 2 days after pCX4 purotsLT virus infection, culture supernatant containing virus obtained by infecting PLT-E cells with v-Abl expression vector (pCX v-Abl GFP) in the same manner as above. After concentration, the cells were infected.
（５）サイトカイン存在下での培養
tsLTとv-Ablを導入した細胞を、サイトカイン非存在下、IL-3存在下、IL-6存在下、IL-7存在下、あるいはIL-3、IL-6およびIL-7の存在下で、33.5℃、5容量％CO2条件で培養し、増殖の状況を観察した。(5) Culture in the presence of cytokines
Cells introduced with tsLT and v-Abl in the absence of cytokines, in the presence of IL-3, in the presence of IL-6, in the presence of IL-7, or in the presence of IL-3, IL-6 and IL-7 The cells were cultured under conditions of 33.5 ° C. and 5% by volume CO 2 , and the state of proliferation was observed.
結果を表１に示す。
The results are shown in Table 1.
表中の○印は増殖が確認されたことを、△は細胞数が維持されたことを、×印は増殖及び維持が確認されなかったことを示している。 In the table, ○ indicates that proliferation was confirmed, Δ indicates that the number of cells was maintained, and X indicates that proliferation and maintenance were not confirmed.
約2ヵ月後、IL-7の存在下で、tsLTとv-Ablを導入した細胞が浮遊細胞として増殖していることが確認された。IL-3、IL-6、及びIL-7の全てを添加した条件下では、tsLT単独、もしくはtsLTとv-Ablを導入した場合に接着細胞の増殖が確認された。IL-3単独、もしくはIL-3、IL-6およびIL-7の全てを添加し、v-Abl単独を導入した条件下では細胞数は維持されたものの、増殖は確認できなかった。また、結果は載せていないがtsLT、v-Ablを導入しない条件下では細胞の維持及び増殖は確認されなかった。 About 2 months later, it was confirmed that cells into which tsLT and v-Abl were introduced proliferated as floating cells in the presence of IL-7. Under the conditions where all of IL-3, IL-6, and IL-7 were added, proliferation of adherent cells was confirmed when tsLT alone or tsLT and v-Abl were introduced. Although the number of cells was maintained under conditions where IL-3 alone or all of IL-3, IL-6 and IL-7 were added and v-Abl alone was introduced, proliferation was not confirmed. In addition, although the results are not listed, maintenance and proliferation of the cells were not confirmed under conditions where tsLT and v-Abl were not introduced.
未分化Ｂ細胞は浮遊細胞であるため、IL-7, tsLT, v-Ablの条件下で増殖してきた浮遊細胞群に含まれる可能性が示唆された。このため、以下、この条件で増殖した細胞群の解析を行うこととした。 Since undifferentiated B cells are floating cells, it was suggested that they may be included in the floating cell group that proliferated under the conditions of IL-7, tsLT, and v-Abl. For this reason, hereinafter, it was decided to analyze the group of cells grown under these conditions.
実験例２ 不死化細胞の表面抗原の解析
IL-7の存在下でtsLTおよびv-Ablを発現させた条件で不死化された細胞の表面抗原をFACScan（BD Biosciences)により解析した。解析した表面抗原は、Ｂ細胞でそれぞれの分化段階を通して広く発現し、Ｂ細胞のマーカーとして汎用されているB220、プロＢ細胞〜プレＢ細胞で発現することが知られているCD25、免疫グロブリン遺伝子の再構成が完了したプレＢ細胞以降で発現が見られるIgMである。これらの各表面抗原を解析するために、抗体として、B220用にPerCP-conjugated anti-mouse CD45R/B220 抗体(BD Biosciences)、CD25用にPE-Cy5-conjugated anti-mouse CD25抗体（eBioscience）、IgM 用にPE-conjugated anti-mouse IgM 抗体（BD Biosciences）を用いた。また、不死化された細胞がv-Ablを発現しているかを確認するために、GFPの発現を調べた。結果を図１に示す。 Experimental Example 2 Analysis of surface antigen of immortalized cells
Surface antigens of cells immortalized under conditions where tsLT and v-Abl were expressed in the presence of IL-7 were analyzed by FACScan (BD Biosciences). The analyzed surface antigen is widely expressed in B cells through each differentiation stage, and is widely used as a marker for B cells. B220, CD25 known to be expressed in pro-B cells to pre-B cells, immunoglobulin genes It is IgM whose expression is seen after the pre-B cell in which reconstitution of is completed. To analyze each of these surface antigens, PerCP-conjugated anti-mouse CD45R / B220 antibody (BD Biosciences) for B220, PE-Cy5-conjugated anti-mouse CD25 antibody (eBioscience) for Ig25, IgM PE-conjugated anti-mouse IgM antibody (BD Biosciences) was used. In order to confirm whether the immortalized cells express v-Abl, the expression of GFP was examined. The results are shown in FIG.
解析の結果、GFP陽性かつB220陽性細胞は78.9 容量%（図１ａ）、GFP陽性かつCD25陽性細胞は68.8容量 %（図１ｂ）であった。GFP陽性かつIgM陽性細胞については、ほとんど確認することができなかった（図１ｃ）。また、図中に示していないがGFP発現細胞は83 容量% であった。このことは、増殖してきたほとんどの細胞（83容量％）が、v-Ablを発現していることを示している。これらの結果から、IL-7, tsLT,およびv-Ablの条件下で不死化された細胞群にはＢ細胞系列の細胞が含まれ、その分化段階はプロＢ〜プレＢである可能性が示された（図１参照）。更に、IgMの発現はほとんど見られなかったことから、IL-7, tsLT,およびv-Ablの条件下で不死化された細胞は、遺伝子再構成が完了してIgMが発現する以前の未分化Ｂ細胞（例えば、限定はされないが、プレB細胞を含む）であると考えられた。 As a result of analysis, GFP positive and B220 positive cells were 78.9% by volume (FIG. 1a), and GFP positive and CD25 positive cells were 68.8% by volume (FIG. 1b). Almost no GFP positive and IgM positive cells could be confirmed (FIG. 1c). Further, although not shown in the figure, GFP-expressing cells were 83% by volume. This indicates that most of the proliferating cells (83% by volume) express v-Abl. From these results, the cell group immortalized under the conditions of IL-7, tsLT, and v-Abl includes B cell lineage cells, and the differentiation stage may be pro-B to pre-B. (See FIG. 1). Furthermore, since almost no IgM expression was observed, cells immortalized under IL-7, tsLT, and v-Abl conditions were undifferentiated prior to completion of gene rearrangement and expression of IgM. It was considered to be a B cell (eg, including but not limited to pre-B cells).
実験例３ AT1細胞の温度依存性増殖とtsLTおよびv-Ablの発現確認
（１）AT1細胞の調製
IL-7, tsLT, v-Ablの条件で不死化された細胞群は、その大部分が大きさの一様な浮遊細胞であったものの、大きさの異なる接着系細胞や浮遊細胞も含まれていた。このため、同様の大きさと内部構造を有する細胞をFACS vantage（BD Biosciences)を利用して、Forward scatterとSide scatterで展開したのち選択し、更にv-Ablを発現する細胞を、GFPを指標として分取した。このv-Ablを発現する細胞を4μg/mlのpuromycinで処理したところ、細胞が死ななかったことから、当該細胞は同時にtsLTを発現している可能性も示唆された。この分取し、且つ薬剤選択を行った細胞群を、「AT1細胞」と名づけ、以下の実験を行った。 Experimental Example 3 Temperature-dependent growth of AT1 cells and confirmation of tsLT and v-Abl expression (1) Preparation of AT1 cells
Most of the cells that were immortalized under the conditions of IL-7, tsLT, and v-Abl were floating cells with a uniform size, but also included adherent cells and floating cells with different sizes. It was. For this reason, cells with the same size and internal structure were selected using FACS vantage (BD Biosciences) and expanded with forward scatter and side scatter, and cells expressing v-Abl were selected using GFP as an indicator. Sorted. When cells expressing this v-Abl were treated with 4 μg / ml puromycin, the cells did not die, suggesting that the cells may also express tsLT at the same time. The cell group from which this sorting and drug selection were performed was named “AT1 cell”, and the following experiment was performed.
(２）AT1細胞の温度依存性増殖
AT1細胞が温度依存的に増殖を行うかについて調べた。具体的には、105個のAT1細胞を12 well(22mm)のプレートに播種し、33.5℃ で培養した。24時間後、温度を33.5℃から39℃へシフトさせた。温度シフトさせた地点を0時間とし、24時間ごとに細胞数を数え（24時目、48時間目、72時間目）、３回の独立した実験の平均値を求めた。結果を図２ａに示す。図中、●は、33.5℃で継続的に培養した場合の細胞数の増加を、また▲は33.5℃から39℃にシフトし72時間培養した場合の細胞数の増加を示す。 なお、エラーバーは標準偏差で示してある。 図2aに示すように、33.5℃では細胞は安定に増殖するのに対し、39℃に温度をシフトすると細胞の増殖が抑えられた。これは、AT1細胞の増殖が、温度感受性tsLTの発現に依存していることを示唆している。(2) Temperature-dependent growth of AT1 cells
It was investigated whether AT1 cells proliferate in a temperature-dependent manner. Specifically, 10 5 AT1 cells were seeded on a 12-well (22 mm) plate and cultured at 33.5 ° C. After 24 hours, the temperature was shifted from 33.5 ° C to 39 ° C. The temperature-shifted point was defined as 0 hour, and the number of cells was counted every 24 hours (24 hours, 48 hours, 72 hours), and the average value of three independent experiments was determined. The result is shown in FIG. In the figure, ● indicates an increase in the number of cells when continuously cultured at 33.5 ° C., and ▲ indicates an increase in the number of cells when shifted from 33.5 ° C. to 39 ° C. and cultured for 72 hours. Error bars are indicated by standard deviation. As shown in FIG. 2a, the cells grew stably at 33.5 ° C., whereas the cell growth was suppressed when the temperature was shifted to 39 ° C. This suggests that AT1 cell proliferation is dependent on the expression of temperature sensitive tsLT.
次に、これらのAT1細胞が実際にtsLT、およびv-Ablを発現しているかをウエスタンブロットにより調べた。特にtsLTに関しては、さらにLTの発現が温度依存的に減少しているかについても調べた。具体的には、33.5℃で培養した細胞（BM(33.5℃)）、39℃で培養して24時間後の細胞（BM(39℃)24hr）、またv-Ablによって癌化したB細胞株であるAbe8（ABE-8）を、v-Abl発現のポジティブコントロールとし、それぞれの細胞について各種タンパク質（v-Abl、c-Abl、Large T、β-Actin）の発現を調べた。セミコンフルエントの細胞をlysis buffer (1容量% Triton X-100, 10 mM Tris-HCl pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 20 mM β-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 20 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride)に溶解し、BCA protein assay reagent (Pierce)によりタンパク質量を定量し、10μｇを10% SDS-PAGEで電気泳動を行った。 Next, it was examined by Western blot whether these AT1 cells actually express tsLT and v-Abl. In particular, regarding tsLT, it was further investigated whether the expression of LT decreased in a temperature-dependent manner. Specifically, cells cultured at 33.5 ° C (BM (33.5 ° C)), cells cultured at 39 ° C 24 hours later (BM (39 ° C) 24hr), and B cell lines transformed into cancer by v-Abl Abe8 (ABE-8) was used as a positive control for v-Abl expression, and the expression of various proteins (v-Abl, c-Abl, Large T, β-Actin) was examined for each cell. Semiconfluent cells were lysed with lysis buffer (1 vol% Triton X-100, 10 mM Tris-HCl pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 20 mM β-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 20 μg / ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) were quantified with BCA protein assay reagent (Pierce), and 10 μg was electrophoresed on 10% SDS-PAGE. .
30分のブロッキングの後、１次抗体（anti-Abl抗体(8E9; Pharmingen)、anti-LT抗体(pAB101; Pharmingen)、anti-β-Actin抗体(Ac-74; Sigma)）を終夜で反応させた。なお、使用した抗体のうち、anti-Abl抗体(8E9; Pharmingen )とanti-LT抗体(pAB101; Pharmingen )は5%スキムミルク-TBSTに希釈し、anti-β-Actin抗体(Ac-74; Sigma)は、5% BSAに希釈して用いた。許容温度である33.5 ℃で培養した細胞は、tsLTの発現が確認されたものの、非許容温度の39.0℃で培養した細胞では、tsLTの発現が有意に減少していた（図 2b、2段目）。v-Ablは、33.5℃、39.0℃ともに発現しており、細胞株であるAbe8と同等のv-Ablを発現していることが分かった。（図 2b、1段目）。 なお、１段目の下のバンドは、内在性のc-Ablである。また、β-Actinは、それぞれの細胞において等しく発現していることが確認された（図 2b、3段目）。以上の結果は、AT1細胞がtsLTとv-Ablの両方の発現によって不死化していることを示している。 After blocking for 30 minutes, the primary antibodies (anti-Abl antibody (8E9; Pharmingen), anti-LT antibody (pAB101; Pharmingen), anti-β-Actin antibody (Ac-74; Sigma)) were reacted overnight. It was. Of the antibodies used, anti-Abl antibody (8E9; Pharmingen) and anti-LT antibody (pAB101; Pharmingen) were diluted in 5% skim milk-TBST and anti-β-Actin antibody (Ac-74; Sigma) Was diluted in 5% BSA. Although tsLT expression was confirmed in cells cultured at the permissive temperature of 33.5 ° C, tsLT expression was significantly decreased in cells cultured at the non-permissive temperature of 39.0 ° C (Figure 2b, 2nd stage). ). v-Abl was expressed at both 33.5 ° C and 39.0 ° C, indicating that v-Abl was expressed equivalent to the cell line Abe8. (Figure 2b, first row). The lower band in the first row is endogenous c-Abl. It was also confirmed that β-Actin was equally expressed in each cell (Fig. 2b, 3rd stage). The above results indicate that AT1 cells are immortalized by the expression of both tsLT and v-Abl.
実験例４ AT1細胞のIL-7依存性
次にAT1細胞のIL-7依存性について調べた。具体的には、まず105個のAT1細胞を12well(22mm)のプレートに播種し、33.5℃でIL-7有り、またはIL-7無しの条件で培養を行った（33.5℃）。培養開始後、24時間毎に4日間にわたって細胞数の計測を行い、３回の独立した実験の平均値を求めた。結果を図３に示す。図中、●はIL-7存在下で培養した場合のAT1細胞数の増加（IL-7(＋)）を、また▲はIL-7非存在下で培養した場合のAT1細胞数の増加IL-7(−)を示している。なお、エラーバーは標準偏差で示してある。図３に示す通り、IL-7の有無によるAT1細胞の増殖に、有意な差は認められなかった。このことから、不死化AT1細胞の増殖は、IL-7に非依存的である可能性が示唆された。IL-7の非存在下で、tsLTとv-Ablを発現させても、増殖する浮遊細胞が見られなかったことから（表１）、IL-7は未成熟なB細胞を不死化する初期には必要であるが、不死化した細胞の維持には必要ではないことが示唆された。 Experimental Example 4 IL-7 dependency of AT1 cells Next, IL-7 dependency of AT1 cells was examined. Specifically, 10 5 AT1 cells were first seeded on a 12-well (22 mm) plate and cultured at 33.5 ° C. with or without IL-7 (33.5 ° C.). After the start of culture, the number of cells was counted every 24 hours for 4 days, and the average value of three independent experiments was determined. The results are shown in FIG. In the figure, ● indicates an increase in the number of AT1 cells (IL-7 (+)) when cultured in the presence of IL-7, and ▲ indicates an increase in the number of AT1 cells when cultured in the absence of IL-7. -7 (-) is shown. Error bars are indicated by standard deviation. As shown in FIG. 3, there was no significant difference in the growth of AT1 cells with or without IL-7. This suggested that the growth of immortalized AT1 cells may be independent of IL-7. Even when tsLT and v-Abl were expressed in the absence of IL-7, no proliferating floating cells were observed (Table 1), so IL-7 immortalizes immature B cells. This suggests that it is necessary for the maintenance of immortalized cells.
実験例５ AT1細胞のin vitroにおける分化能の検討
AT1細胞を用いて抗体を産生させることが可能かどうかを調べるために、in vitroでIgM発現誘導を試みた。まず2 x 105個の細胞を、35-mmプレートに播種し、IL-7存在下、33.5℃で、以下の条件で培養を行った。 Experimental Example 5 Examination of in vitro differentiation potential of AT1 cells
In order to examine whether it is possible to produce antibodies using AT1 cells, we attempted to induce IgM expression in vitro. First, 2 × 10 5 cells were seeded on a 35-mm plate and cultured in the presence of IL-7 at 33.5 ° C. under the following conditions.
（a） 骨髄（BM）由来の間葉系細胞（BM stroma; p53の不活性化により不死化している）との共培養。
（b） 成熟B細胞への分化に関与するとされるIL-6の存在下での培養。
（c） Trichostatin A（Wako）（Seo et al., 2005；参考文献４４）の存在下での培養。
（d） IL-6とTrichostatin Aの存在下での培養。(A) Co-culture with bone marrow (BM) -derived mesenchymal cells (BM stroma; immortalized by inactivation of p53).
(B) Culture in the presence of IL-6, which is considered to be involved in differentiation into mature B cells.
(C) Culture in the presence of Trichostatin A (Wako) (Seo et al., 2005; reference 44).
(D) Culture in the presence of IL-6 and Trichostatin A.
IL-6については、サイトカイン過剰発現細胞（WI-6：東京医科歯科大の烏山先生より分与）を3日間培養した後の上清をコンデイションドメディウムとして、これを最終濃度が培養液の5容量%になるように加えた。また、Trichostatin Aについては2.5 ng/ml となるように添加した。 For IL-6, the supernatant after culturing cytokine-overexpressing cells (WI-6: provided by Prof. Kashiyama of Tokyo Medical and Dental University) for 3 days was used as a conditioned medium. Added to 5% by volume. Trichostatin A was added at 2.5 ng / ml.
培養開始3週間後に、細胞をPerCP-conjugated anti-mouse CD45R/B220 抗体（BD Biosciences）、PE-conjugated anti-mouse IgM 抗体（BD Biosciences）により染色を行い、B220,およびIgMの発現変化をFACScanにより調べた。結果を図４に示す。 Three weeks after the start of culture, cells were stained with PerCP-conjugated anti-mouse CD45R / B220 antibody (BD Biosciences) and PE-conjugated anti-mouse IgM antibody (BD Biosciences), and B220 and IgM expression changes were detected with FACScan. Examined. The results are shown in FIG.
図４からわかるように、B220に関しては、Trichostatin Aを加えることで、B220陰性の細胞が増殖しているように思われたが、IgMに関しては、全ての条件において顕著な変化は確認されなかった。従って、in vitroでのAT1細胞からIgM発現細胞への分化条件は、更に検討する必要がある。 As can be seen from FIG. 4, B220-negative cells seemed to grow by adding Trichostatin A for B220, but for IgM, no significant change was observed in all conditions. . Therefore, the differentiation conditions from AT1 cells to IgM-expressing cells in vitro need to be further investigated.
実験例６ AT1細胞のNOD/SCIDマウスを用いた分化能の検討
AT1細胞をマウス個体に移植した際に分化が進むかどうかを、4週齢メスのNOD/SCID マウス（日本クレア）を用いて調べた。細胞は、ficol（アマシャム）を使って白血球画分を分離した。NOD/SCID マウスは、成熟B細胞、成熟T細胞が存在しないことが知られている。このため、IgM陽性かつGFP陽性の細胞が確認できれば、移植した細胞の分化が進んだものと考えられる。 Experimental Example 6 Examination of differentiation potential of AT1 cells using NOD / SCID mice
Whether AT1 cells were differentiated when transplanted into mice was examined using 4-week-old female NOD / SCID mice (Claire Japan). Cells separated the leukocyte fraction using ficol (Amersham). NOD / SCID mice are known not to have mature B cells or mature T cells. For this reason, if the IgM positive and GFP positive cells can be confirmed, it is considered that the transplanted cells have been differentiated.
具体的には、200μlのPBSに懸濁した2×106個のAT1細胞を、マウスの尾部から静脈中に移植注射し、2〜3週間飼育を行った。なお、移植しない個体をネガティブコントロールとして用いた。AT1細胞を移植した個体は移植後2週間目から動きに異常をきたすようになり、移植後約3週間で死亡した。(3匹/3匹)。このことは、移植を受けた個体中でAT1細胞が定着、増殖している可能性を示唆している。Specifically, 2 × 10 6 AT1 cells suspended in 200 μl of PBS were transplanted and injected into the vein from the tail of the mouse and reared for 2 to 3 weeks. Individuals not transplanted were used as negative controls. Individuals transplanted with AT1 cells became abnormal in movement from the second week after transplantation and died about three weeks after transplantation. (3/3). This suggests that AT1 cells may have established and proliferated in the transplanted individual.
AT1細胞を移植した個体について、移植2週間後の骨髄（BM）及び脾臓（spleen）中の骨髄細胞を採取し、PE-conjugated anti-mouse IgM 抗体（BD Biosciences）により染色後、FACScanにより解析をおこなった。解析の結果、GFPを発現する細胞が確認され、移植したAT1細胞がマウス個体中で定着、増殖していることが示された。また、in vitroで継代維持、もしくは分化誘導した際にはほとんど確認することができなかったGFP陽性かつIgM陽性の細胞が（図１、図４）、骨髄由来の白血球画分、並びに脾臓由来の白血球画分に共に確認された(図５ｂ, ｄ)。一方の、コントロールとして用いたマウスからは、GFP陽性かつIgM陽性の細胞が確認できなかったことから(図５ａ,ｃ)、移植したAT1細胞がマウス個体中で分化した可能性が強く示唆された。発現細胞の割合は、骨髄では22.5容量％、脾臓では31.6容量％であり、脾臓でより多くのIgM陽性細胞が確認できた。 (図５ｂ, ｄ)。 Bone marrow cells in the bone marrow (BM) and spleen (spleen) 2 weeks after transplantation are collected from individuals transplanted with AT1 cells, stained with PE-conjugated anti-mouse IgM antibody (BD Biosciences), and analyzed by FACScan I did it. As a result of the analysis, cells expressing GFP were confirmed, and it was shown that the transplanted AT1 cells were established and proliferated in the mouse individual. In addition, GFP-positive and IgM-positive cells that could hardly be confirmed when maintained in vitro or induced for differentiation (Figs. 1 and 4), leukocyte fraction derived from bone marrow, and spleen Together with the leukocyte fraction (Fig. 5b, d). On the other hand, since GFP-positive and IgM-positive cells could not be confirmed from the mouse used as a control (FIGS. 5a and 5c), it was strongly suggested that the transplanted AT1 cells were differentiated in the mouse individual. . The ratio of the expressed cells was 22.5% by volume in the bone marrow and 31.6% by volume in the spleen, and more IgM positive cells were confirmed in the spleen. (Fig. 5b, d).
以上の結果から、IL-7, tsLT, v-Ablの条件で、不死化されたAT1細胞が得られ、このAT1細胞はNOD/SCIDマウス体内でIgM発現細胞へと分化する能力を有することが示唆された。 From the above results, immortalized AT1 cells were obtained under the conditions of IL-7, tsLT, and v-Abl, and these AT1 cells have the ability to differentiate into IgM-expressing cells in NOD / SCID mice. It was suggested.
実験例７ AT2細胞における不死化細胞の表面抗原の解析
AT1細胞を不死化した条件と同じ条件であるIL-7の存在下で、tsLTではなくLTとv-Ablを発現させた条件で不死化された細胞の表面抗原をFACScan （BDBiosciences)により解析した。解析した表面抗原はB細胞でそれぞれの分化段階を通して広く発現し、B細胞のマーカーとして汎用されているB220、プロB〜プレB細胞で発現することが知られているCD25、免疫グロブリン遺伝子の再構成が完了したプレB細胞以降で発現が見られるIgMである。それぞれを解析するために用いた抗体はPerCP-conjugated anti-mouse CD45R/B220 抗体(BD Biosciences)、PE-Cy5-conjugated anti-mouse CD25抗体（eBioscience）、PerCP-conjugated anti-mouse IgM 抗体（BD Biosciences）である。 Experimental Example 7 Analysis of surface antigens of immortalized cells in AT2 cells
FACScan (BDBiosciences) analyzed the surface antigens of cells immortalized under the conditions that expressed LT and v-Abl instead of tsLT in the presence of IL-7, which was the same as the conditions under which AT1 cells were immortalized. . The analyzed surface antigens are widely expressed in B cells throughout their differentiation stages, and are widely used as B cell markers, B220, CD25, which is known to be expressed in pro-B to pre-B cells, It is IgM whose expression is seen after pre-B cells whose construction is completed. The antibodies used for the analysis were PerCP-conjugated anti-mouse CD45R / B220 antibody (BD Biosciences), PE-Cy5-conjugated anti-mouse CD25 antibody (eBioscience), PerCP-conjugated anti-mouse IgM antibody (BD Biosciences). ).
また、不死化された細胞がv-Ablを発現しているかを確認するために、マーカーとして用いられたGFPの発現も調べた。 In addition, in order to confirm whether the immortalized cells express v-Abl, the expression of GFP used as a marker was also examined.
その解析の結果、GFP陽性かつB220陽性細胞は70.7容量 %（図６-a）、GFP陽性かつCD25陽性細胞は53.6容量 %（図６-b）であった。GFP陽性かつIgM陽性細胞については、ほとんど確認することができなかった（図６-c）。また、図中に示していないがGFP発現細胞は93.2容量 % であった。この事は、増殖してきたほとんどの細胞がv-Ablを発現していることを示唆している。 As a result of the analysis, GFP positive and B220 positive cells were 70.7% by volume (FIG. 6-a), and GFP positive and CD25 positive cells were 53.6% by volume (FIG. 6b). Almost no GFP-positive and IgM-positive cells could be confirmed (FIG. 6-c). Moreover, although not shown in the figure, the GFP-expressing cells were 93.2% by volume. This suggests that most proliferating cells express v-Abl.
以上の結果から、IL-7, LT, v-Ablの条件下で不死化された細胞群においてもB細胞系列の細胞が含まれ、その分化段階はプロB〜プレBである可能性が示唆された。更に、IgMの発現はほとんど見られないことから、遺伝子再構成が起こるPre-B後期以前の細胞が多く含まれる細胞群であると考えられた。この細胞群をAT2細胞とした。 The above results suggest that the cells that are immortalized under the conditions of IL-7, LT, and v-Abl also contain B cell lineage cells, and their differentiation stage may be pro-B to pre-B It was done. Furthermore, since almost no IgM expression was observed, it was considered to be a cell group containing many cells before the Pre-B late stage in which gene rearrangement occurs. This cell group was designated as AT2 cells.
実験例８ AT２細胞のNOD/SCIDマウスを用いた分化能の検討
AT2細胞をマウス個体に移植した際に分化が進むかどうかを、4週齢メスのNOD/SCID マウス（日本クレア）を用いて調べた。NOD/SCID マウスは、成熟B細胞、成熟T細胞が存在しないことが知られており、IgM陽性かつGFP陽性の細胞が確認できれば、移植した細胞の分化が進んだと考えられる。 Experimental Example 8 Examination of differentiation potential of AT2 cells using NOD / SCID mice
Whether AT2 cells were differentiated when transplanted into mice was examined using 4-week-old female NOD / SCID mice (Claire Japan). NOD / SCID mice are known to have no mature B cells or mature T cells. If IgM-positive and GFP-positive cells can be confirmed, differentiation of the transplanted cells is considered to have progressed.
200μlのPBSに懸濁した2×106個の細胞を、マウスの尾部から静脈中に移植注射した。移植したマウスは2〜3週間飼育を行った。定着した細胞の分化段階が進みIgMの発現が見られる可能性を考え、移植2週間後の骨髄及び脾臓中の骨髄細胞を採取し、PerCP-conjugated anti-mouse IgM 抗体（BD Biosciences）により染色後、FACScanにより解析をおこなった。2 × 10 6 cells suspended in 200 μl of PBS were implanted and injected into the vein from the tail of the mouse. The transplanted mice were raised for 2-3 weeks. Taking into consideration the possibility that IgM expression may be observed as the differentiation stage of established cells progresses, bone marrow and spleen bone marrow cells are collected 2 weeks after transplantation and stained with PerCP-conjugated anti-mouse IgM antibody (BD Biosciences) Analysis was performed by FACScan.
その解析の結果、GFPを発現する細胞が確認され、移植したAT2細胞がマウス個体中で定着、増殖していることが示された。また、in vitroで継代維持、もしくは分化誘導した際にはほとんど確認することができなかったGFP陽性かつIgM陽性の細胞が（図６）、骨髄、脾臓由来の白血球画分で共に確かめられた(図７a, b)。このことから、AT2細胞においても、AT1細胞と同じく、移植したAT2細胞が分化した可能性が強く示唆された。 As a result of the analysis, cells expressing GFP were confirmed, and it was shown that the transplanted AT2 cells were established and proliferated in the mouse individual. In addition, GFP-positive and IgM-positive cells that could hardly be confirmed when subcultured or differentiated in vitro (Fig. 6) were confirmed in the leukocyte fractions derived from bone marrow and spleen. (Fig. 7a, b). This strongly suggested the possibility that the transplanted AT2 cells were differentiated in AT2 cells as well as AT1 cells.
そのマウス個体中での、発現細胞の割合は、骨髄では24.2容量 %、脾臓では19.1容量 % であり、骨髄でより多くのIgM陽性細胞が確認できた。 The percentage of expressed cells in the mouse was 24.2% by volume in the bone marrow and 19.1% by volume in the spleen, and more IgM positive cells were confirmed in the bone marrow.
以上の結果をまとめると、AT2細胞はIgMを発現するB細胞へと分化する能力を有していることが示唆された。 Summarizing the above results, it was suggested that AT2 cells have the ability to differentiate into B cells expressing IgM.
なお、本明細書中に引用する参考文献は下記の通りであり、その内容は本願明細書の一部として援用される。 References cited in the present specification are as follows, and the contents thereof are incorporated as a part of the present specification.
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13. Jat and Sharp, 1986, Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts. J. Virol. 59: 746-50.
14. Tegtmeyer and Ozer, 1971, Temperature-sensitive mutants of simian virus 40:
infection of permissive cells. J. Virol. 8: 516-24.
15. Ray et al., 1992, Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen. J. Virol. 66: 6509-16.
16. Jat and Sharp, 1989, Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature
17. Jat et al., 1991, Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA. 88: 5096-100.
18. Obinata, 2001, Possible applications of conditionally immortalized tissue cell lines with differentiation functions. Biochem. Biophys. Res. Commun. 286: 667-72.
19. O’Hare et al., 2001, Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells. Proc. Natl. Acad. Sci. USA. 98: 646-51
20. Namen et al., 1988, . Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line. J Exp Med. Apr 1;171(4):1073-89.
21. Grabstein et al., 1993, Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody. J. Exp. Med. 178: 257-64.
22. von Freeden-Jeffry et al., 1995, Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-26.51. Rosenberg, 1994, Abl-mediated transformation, immunoglobulin gene rearrangements and arrest of B lymphocyte differentiation. Semin. Cancer Biol. 5: 95-102.
23. Jackson and Baltimore, 1989, N-terminal mutations activate the leukemogenic
potential of the myristoylated form of c-abl. EMBO J. 8: 449-56
24. Martensson and Ceredig, 2000, Role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development. Immunology 101,435-441.
25. Wong and Witte, 2004, The BCR-ABL story: bench to bedside and back. Annu. Rev. Immunol.22: 247-306.
26. Druker et al., 1996, Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2: 561-6.
27. Nagar et al., 2002, Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571).Cancer Res.;62(15):4236-43.
28. Weisberg et al., 2005, Characterization of AMN107, a selective inhibitor of
native and mutant Bcr-Abl. Cancer Cell.;7(2):129-41.
29. Shah et al., 2004, Overriding imatinib resistance with a novel ABL kinase inhibitor. Science;305(5682):399-401.
30. Gumireddy et al., 2005, A non-ATP-competitive inhibitor of BCR-ABL overrides imatinib resistance. Proc Natl Acad Sci U S A.;102(6):1992-7.
31. Wolff et al., 2005, PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia. Blood.;105(10):3995-4003.
32. O’Hare et al., 2004, Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML.Blood. ;104(8):2532-9.
33. Radfar et al., 1998, . p19(Arf) induces p53-dependent apoptosis during abelson virus-mediated pre-B cell transformation. Proc. Natl. Acad. Sci. USA. 95: 13194-9.
34. Jat and Sharp, 1989, . Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.Mol. and Cell. Boil. 9, 1672-1681.
35. O’Hare et al., 2001, Conditional immortalization of freshly isolated human
mammary fibroblasts and endothelial cells. Proc. Natl. Acad. Sci. USA. 98: 646-51
36. Morgenstern and Land, 1990, Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res.;18(12):3587-96.
37. Onishi et al., 1996, Applications of retrovirus-mediated expression cloning. Exp Hematol.;24(2):324-9.
38. Akagi et al., 2000, v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc Natl Acad Sci U S A.;97(13):7290-5.
39. Akagi et al., 2003, Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation.Proc Natl Acad Sci U S A. 100(23):13567-72
40. Hawley et al., 1994, Versatile retroviral vectors for potential use in gene therapy. Gene Ther.;1(2):136-8.
41. Halene et al., 1999, Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector.Blood.;94(10):3349-57.
42. Riviere et al., 1995, Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci U S A.;92(15):6733-7.
43. Cerutti，A. et al., 1998, CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line. J. Immunol. 160: 2145-2157.
44. Seo, H., et al., 2005, Rapid generation of specific antibodies by enhanced homologous recombination. Nat. Biotechnol. 23: 731-735. References
1. Seo, H., et al., 2005, Rapid generation of specific antibodies by enhanced homologous recombination. Nat. Biotechnol. 23: 731-735.
2. Abelson and Rabstein, 1970, Lymphosarcoma: virus-induced thymic-independent disease in mice.Cancer Res. 30: 2213-22.
3. Goff et al., 1982, Chromosomal assignment of the endogenous proto-oncogene C-abl. Science. 218: 1317-9.
4. Hantschel and Superti-furga, 2004, Regulation of the c-Abl and Bcr-Abl tyrosine kinases. Nat. Rev. Mol. Cell Biol. 5: 33-44.
5. Shore et al., 2002, Transforming pathways activated by the v-Abl tyrosine kinase. Oncogene. 21: 8568-76.
6. Jackson and Baltimore, 1989, N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl.EMBO J. 8: 449-56
7. Rosenberg, 1994, Abl-mediated transformation, immunoglobulin gene rearrangements and arrest of B lymphocyte differentiation. Semin. Cancer Biol. 5: 95-102
8. Druker et al., 1996, Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2: 561-6.
9. Muljo and Schlissel, 2003, A small molecule Abl kinase inhibitor induces differentiation of Abelson virus-transformed pre-B cell lines. Nat. Immunol. 4: 31-7.
10. Graham et al., 1975, Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and
11. Rundell and Parakati, 2001, The role of the SV40 ST antigen in cell growth promotion and transformation.Semin. Cancer Biol. 11: 5-13.
12. Kamijo et al., 1997, Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell. 91: 649-59.
13. Jat and Sharp, 1986, Large T antigens of
14. Tegtmeyer and Ozer, 1971, Temperature-sensitive mutants of simian virus 40:
infection of permissive cells. J. Virol. 8: 516-24.
15. Ray et al., 1992, Functional characterization of temperature-sensitive mutants of
16. Jat and Sharp, 1989, Cell lines established by a temperature-sensitive
17. Jat et al., 1991, Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.Proc. Natl. Acad. Sci. USA. 88: 5096-100.
18. Obinata, 2001, Possible applications of conditionally immortalized tissue cell lines with differentiation functions. Biochem. Biophys. Res. Commun. 286: 667-72.
19. O'Hare et al., 2001, Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.Proc. Natl. Acad. Sci. USA. 98: 646-51
20. Namen et al., 1988,. Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line. J Exp Med. Apr 1; 171 (4): 1073-89.
21. Grabstein et al., 1993, Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody. J. Exp. Med. 178: 257-64.
22. von Freeden-Jeffry et al., 1995, Lymphopenia in interleukin (IL) -7 gene-deleted mice identify IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-26.51. Rosenberg, 1994, Abl -mediated transformation, immunoglobulin gene rearrangements and arrest of B lymphocyte differentiation.Semin. Cancer Biol. 5: 95-102.
23. Jackson and Baltimore, 1989, N-terminal mutations activate the leukemogenic
potential of the myristoylated form of c-abl.EMBO J. 8: 449-56
24. Martensson and Ceredig, 2000, Role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development. Immunology 101,435-441.
25. Wong and Witte, 2004, The BCR-ABL story: bench to bedside and back. Annu. Rev. Immunol. 22: 247-306.
26. Druker et al., 1996, Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2: 561-6.
27. Nagar et al., 2002, Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res.; 62 (15): 4236-43.
28. Weisberg et al., 2005, Characterization of AMN107, a selective inhibitor of
native and mutant Bcr-Abl.Cancer Cell.; 7 (2): 129-41.
29. Shah et al., 2004, Overriding imatinib resistance with a novel ABL kinase inhibitor. Science; 305 (5682): 399-401.
30. Gumireddy et al., 2005, A non-ATP-competitive inhibitor of BCR-ABL overrides imatinib resistance.Proc Natl Acad Sci US A.; 102 (6): 1992-7.
31.Wolf et al., 2005, PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia. Blood.; 105 (10): 3995-4003.
32. O'Hare et al., 2004, Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML.Blood.; 104 (8): 2532-9.
33.Radfar et al., 1998, .p19 (Arf) induces p53-dependent apoptosis during abelson virus-mediated pre-B cell transformation.Proc. Natl. Acad. Sci. USA. 95: 13194-9.
34. Jat and Sharp, 1989, .Cell lines established by a temperature-sensitive
35. O'Hare et al., 2001, Conditional immortalization of freshly isolated human
mammary fibroblasts and endothelial cells. Proc. Natl. Acad. Sci. USA. 98: 646-51
36. Morgenstern and Land, 1990, Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.Nucleic Acids Res.; 18 (12): 3587-96.
37. Onishi et al., 1996, Applications of retrovirus-mediated expression cloning.Exp Hematol .; 24 (2): 324-9.
38. Akagi et al., 2000, v-Crk activates the phosphoinositide 3-kinase / AKT pathway in transformation.Proc Natl Acad Sci US A.; 97 (13): 7290-5.
39. Akagi et al., 2003, Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation.Proc Natl Acad Sci US A. 100 (23): 13567-72
40. Hawley et al., 1994, Versatile retroviral vectors for potential use in gene therapy.Gene Ther.; 1 (2): 136-8.
41. Halene et al., 1999, Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector.Blood.; 94 (10): 3349-57.
42. Riviere et al., 1995, Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.Proc Natl Acad Sci US A.; 92 (15): 6733-7.
43. Cerutti, A. et al., 1998, CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM + IgD + B cell line. J. Immunol. 160 : 2145-2157.
44. Seo, H., et al., 2005, Rapid generation of specific antibodies by enhanced homologous recombination. Nat. Biotechnol. 23: 731-735.
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