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iPS/ES CELL-SPECIFIC ANTIBODY HAVING CYTOTOXICITY TO TARGET CELLS AND USE THEREOF meetings

Foreign code F150008135
Posted date Feb 13, 2015
Country WIPO
International application number 2013JP084374
International publication number WO 2014098243
Date of international filing Dec 20, 2013
Date of international publication Jun 26, 2014
Priority data
  • P2012-280259 (Dec 21, 2012) JP
Title iPS/ES CELL-SPECIFIC ANTIBODY HAVING CYTOTOXICITY TO TARGET CELLS AND USE THEREOF meetings
Abstract Provided are: a monoclonal antibody capable of recognizing a lipid substance that is present on the surface of iPS and ES cells as an epitope and not recognizing EC cells; the aforesaid antibody having a cytotoxicity to target cells; a method for preparing a uniform mass of differentiated cells containing no undifferentiated cells, said method comprising contacting a mass of cells having been differentiated from iPS or ES cells with the aforesaid antibody and collecting surviving cells; an agent for cell transplantation therapy, said agent comprising a mass of differentiated cells obtained by the aforesaid method, etc.
Outline of related art and contending technology BACKGROUND ART
Human induced pluripotent stem cell (iPS cell) by an established, with the use of a pluripotent stem cell transplantation treatment for the practical application of the door is opened. For example, such as Parkinson's disease or diabetes type I in the case of chronic disease, to establish iPS cells from the patient in the, cells necessary for the induction of differentiated can be autologous to the patient if the, human embryonic stem cells (ES cells) associated with the use of ethical issues (that is, life of the destruction of early embryos can also be said that bud break) or at the time of implantation can be to avoid the problem of rejection. On the other hand, in the establishment of iPS cells to from a 2-3 months at the shortest until induction are necessary, such as spinal cord injury or fulminant hepatitis is a disease that requires an early treatment, various HLA type of iPS cells or differentiated cells derived therefrom and banking, they can be used to be considered in the allograft.
However, such as ES cells or iPS cells such as pluripotent stem cells to differentiate into cardiac nerve cells and are cultured under conditions, differentiated cells remain undifferentiated cells in the population, the cause of tumorigenesis (teratoma, carcinogenesis) and, further iPS cells, initialized to the artificial cells but also causes the problem of safety of a specific (i.e., c-Myc such as proto-oncogene with the introduction of the use of viral vectors by the tumorigenesis of risk, that is from a somatic cell depending on the type of risk such as tumorigenesis by differentiation resistance) also suffers. In this way, a reproducing a pluripotent stem cell to a practical use of the transplantation treatment, which overcomes the problems of tumorigenesis is essential. Cell-derived iPS so far is suppressing of carcinogenesis, cancer gene combinations have reprogramming factor does not include, the use of non-viral vectors, such as a protein by the introduction in the establishment of iPS cells established from the viewpoint of a safer iPS has been made various attempts have been made. However, these are some of the risk of the production of iPS in the suppression of carcinogenesis by devising the indirect approach and is not intended to, can be prevented from being completely is not at a level of carcinogenesis. In addition, a common pluripotent stem cells ES cells but also causes the remaining tumorigenesis by undifferentiated cells (target cells other than many types of cells are mixed to form a teratoma mass) also to a risk, an effective resolution is not provided.
However, sugar chain recognizing antibody is, a change in cell surface sugar chain of the probe and the microtopography of the helmet, human iPS/ES cells are also commonly used as a marker antibody. That is, SSEA3, and SSEA4 of the epitopes comprise glycolipids, TRA-1-60, one of the epitopes comprise TRA-1-81 is keratan sulfate. However, most of these existing antibody, cell EC epitamial (embryonal carcinoma cell, fetal cancer cells) was obtained as an immunogen and, in addition to EC iPS/ES cells also react with the cells (cancer cells) (non-patent document 1). Therefore, stem cell research and regenerative medicine in research, does not react EC cells, an antibody that reacts only with the appearance of iPS/ES cells has been expected. Recently, Choo is, human ES cells is used as an immunogen, and does not react with anti-human ES cell EC cell antibodies (mAb84) is reported (patent document 1), this antibody also reacts to the human iPS cells is not described whether or not (patent document 2).
The inventors of the present invention, human iPS cells (Tic) immunizing a mouse as an immunogen, obtained for hybridomas, human iPS cells and human EC cells by performing differential screening, cell EC iPS/ES cells positive and the negative antibody (R-10 g) were successfully obtained (Patent Document 2). This antibody is, iPS/ES binding proteins on the cell surface, or TRA-1-60 TRA-1-81 is different from the epitope recognize keratan sulfate. However, the cell is a human iPS/ES R-10 g does not have a cytotoxic activity, the antibody, the differentiated cell population remaining in human iPS/ES is used to remove cells, flow cytometry or affinity support separation using operations become necessary.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 iPS及びES細胞表面上の脂質性物質をエピトープとして認識し、かつEC細胞を認識しないモノクローナル抗体。

[請求項2]
 iPS及びES細胞がヒト由来である、請求項1記載の抗体。

[請求項3]
 ハイブリドーマR-17F(受託番号:NITE BP-01425)により産生されるモノクローナル抗体、又は該モノクローナル抗体が認識する脂質性物質の領域と同一の領域をエピトープとして認識するモノクローナル抗体である、請求項1又は2記載の抗体。

[請求項4]
 脂質性物質が糖脂質であり、前記領域が下記一般式:
Fuc-Hex-HexNAc-Hex-Hex
(式中、Fucはフコース、Hexはヘキソース、HexNAcはN-アセチルヘキソサミンを示す。)で表される糖鎖を含む、請求項1~3のいずれか1項に記載の抗体。

[請求項5]
 少なくとも糖脂質中の下記式:
Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc
(式中、Fucはフコース、Galはガラクトース、GlcNAcはN-アセチルグルコサミン、Glcはグルコースを示す。)で表される糖鎖を含む領域をエピトープとして認識する、請求項4記載の抗体。

[請求項6]
 (a)配列番号:1で示されるアミノ酸配列を含むCDR、
(b)配列番号:2で示されるアミノ酸配列を含むCDR、
(c)配列番号:3で示されるアミノ酸配列を含むCDR、
(d)配列番号:4で示されるアミノ酸配列を含むCDR、
(e)配列番号:5で示されるアミノ酸配列を含むCDR、及び
(f)配列番号:6で示されるアミノ酸配列を含むCDR
を含む、請求項1~5のいずれか1項に記載の抗体。

[請求項7]
 (1)配列番号:8に示されるアミノ酸配列を含む重鎖可変領域、及び
(2)配列番号:10に示されるアミノ酸配列を含む軽鎖可変領域
を含む請求項6記載の抗体。

[請求項8]
 標的細胞に対して細胞障害活性を有する、請求項1~7のいずれか1項に記載の抗体。

[請求項9]
 請求項1~8のいずれか1項に記載の抗体を含有してなる、iPS又はES細胞検出用試薬。

[請求項10]
 細胞サンプルを請求項1~8のいずれか1項に記載の抗体と接触させ、該抗体と結合した該サンプル中の細胞を検出することを含む、iPS又はES細胞の検出方法。

[請求項11]
 請求項1~8のいずれか1項に記載の抗体を含有してなる、iPS又はES細胞除去剤。

[請求項12]
 前記抗体に対する二次抗体をさらに含有してなる、請求項11記載の剤。

[請求項13]
 細胞集団を請求項1~8のいずれかに記載の抗体と接触させることを含む、該細胞集団中のiPS又はES細胞の除去方法。

[請求項14]
 細胞集団を、さらに前記抗体に対する二次抗体に接触させることを含む、請求項13記載の方法。

[請求項15]
 iPS又はES細胞から分化させた細胞集団を請求項1~8のいずれか1項に記載の抗体と接触させ、生存する細胞を回収することを含む、未分化細胞を含まない均一な分化細胞集団の作製方法。

[請求項16]
 前記iPS又はES細胞から分化させた細胞集団を、さらに前記抗体に対する二次抗体に接触させることを含む、請求項15記載の方法。

[請求項17]
 iPS又はES細胞から分化させた細胞集団と、請求項1~8のいずれか1項に記載の抗体とを組み合わせてなる、細胞移植療法剤。

[請求項18]
 請求項15又は16記載の方法により得られる分化細胞集団を含有してなる、細胞移植療法剤。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • THE RITSUMEIKAN TRUST
  • NATIONAL INSTITUTE OF BIOMEDICAL INNOVATION
  • Inventor
  • KAWASAKI, Toshisuke
  • KAWASAKI, Nobuko
  • FURUE, Miho
  • KAWABATA, Kenji
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
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