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論文(リポジトリ)
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鈴木, 訓充 ; 岸, 賢治 ; 成田, 美和子 ; 古川, 達雄 ; 高橋, 益広 ; 相澤, 義房
概要:
As an alternative treatment with allogeneic bone marrow transplantation, a possibility of autologous stem cell transplan
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tation for chronic myelogenous leukemia (CML) was studied. The most critical problem in this study is how to purify normal stem cells from autologous bone marrow or peripheral blood cells. Although normal stem cells are present in these patients in chronic phase, absolute amount of these cells are very small and difficult to detect by regular chromosome analysis. Concerning to a previous report, appearance of Philadelphia chromosome (Ph) negative clones was observed after long-term marrow culture (LTMC) of CML patients. Our previous studies had shown that addition of interferon-α (IFNα) in LTMC preferentially decrease Ph cells. In the present study, we investigated the frequencies of leukemic clones in suspending-and adherent-fraction of LTMC by reverse transcriptional-polymerase chain reaction (RT-PCR) for bcr-abl, CML specific and chromosome analysis of a single progenitor derived colony. All colonies derived from suspending cells presented either bcr-abl and Ph chromosome in both cultures with and without IFNα. In all 11 colonies derived from adherent-fraction of LTMC without IFNα, bcr-abl was detectable, whereas 2/2 CFU-mix and 3/11 CFU-GM were negative for bcr-abl from the culture with IFNα. Results from chromosome analysis of single colony was similar to that by RT-PCR. These results indicate the possibility of application of LTMC with IFNα to eliminate leukemic clones from CML marrow cells, however, there remains a problem that stem cell number decreases during LTMC with IFNα. Our investigation of ex vivo expansion for human hematopoietic stem cells is still experimental, and more progression is necessary for clinical application.
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| 2.
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論文(リポジトリ)
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岩谷, 俊平 ; 成田, 美和子 ; 増子, 正義 ; 西澤, 幹則 ; 大岩, 恵理 ; 井田, 桃里 ; 岩渕, 南 ; 内山, 孝由 ; 柴崎, 康彦 ; 瀧澤, 淳 ; 曽根, 博仁 ; 高橋, 益廣
概要:
imatinibと改変型WT1ペプチドワクチンの併用療法を行ったHLA-A*24:02陽性の慢性骨髄性白血病慢性期（chronic myeloid leukemia-chronic phase；CML-CP）の症例に対し，ワクチン接種前から
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４週間毎に14日の末梢血単核球を混合リンパ球ペプチド培養（mixed lymphocyte peptide culture；MLPC）行い，WT1/MHC-tetramer+ CD8+細胞の末梢血CD8陽性細胞中の頻度の変動を６年間継続して解析した。ペプチドを添加した自己B細胞株を標的細胞として細胞傷害活性試験を行い，今回検出されたWT1/MHC-tetramer+CD8+細胞は確実に細胞傷害活性を有することを確認した。WT1ペプチドワクチンの臨床効果も確認された。すなわち，ワクチン投与終了７ヶ月後には，bcr/ablコピー数が次第に減少しMMR（major molecular response；MMR） に入り， その後，CMR（complete molecular response；CMR）に到達し，WT1ペプチドワクチン投与終了後５年経過した現在もCMRは維持されている。WT1ペプチドワクチンとimatinib併用療法は，TKI（tyrosine kinase inhibitor；TKI）が十分に作用できないCML幹細胞に対し有効である可能性が示された。<br />To clarify the effectiveness and safety of WT1 peptide vaccination against the residual CML cells, we started WT1 peptide vaccination in combination with regular dose of imatinib for a CML-CP patient who had been treated with 400 mg imatinib for 4 years but not achieved MMR. HLA-A*24:02-restricted mutant type WT1 peptide vaccination was undertaken 22 times totally. The appearance of WT1-specific CTLs in PB was confirmed by evaluating the frequency of MHC/WT1 tetramer+CD8+ T cells by using mixed lymphocyte peptide culture（MLPC）system every 4 weeks. WT1 tetramer+ cells detected by MLPC system were investigated for WT1 specific cytotoxicity. Bcr-abl transcripts have decreased to less than 500 copies by the administration of WT1 peptides every 4 weeks. After 7 months from the cessation of vaccination, transcripts decreased to the level of CMR, which is lasting thereafter Tumor antigen specific peptide vaccine therapy in combination with molecular targeted therapy is one of the potent methods for eradicating cancer stem cells.
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| 3.
論文(リポジトリ) |
論文(リポジトリ)
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岩谷, 俊平 ; 成田, 美和子 ; 増子, 正義 ; 西澤, 幹則 ; 大岩, 恵理 ; 柴崎, 康彦 ; 内山, 孝由 ; 瀧澤, 淳 ; 曽根, 博仁 ; 高橋, 益廣
概要:
抗原特異的細胞傷害性T細胞(CTL)は抗腫瘍免疫療法において重要な役割を担っている。微量残存CML細胞を根絶するため，imatinib療法を４年間受けたCML症例にWT1ペプチドワクチンを投与した。混合リンパ球ペプチド培養(MLPC)を行い
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，WT1/MHC-tetramer+CD8+細胞の出現頻度の評価によって末梢血中のWT1特異的CTLの増幅効果の推移を確認した。その結果，WT1ペプチドワクチン投与を終了し６年経過した現在も，WT1特異的CTL はCD8陽性細胞中5-15x10^<-6>の頻度で検出され続けていることが確認された。CTLの検出の検討に加えて，長期間増幅されたWT1特異的CTLのT細胞受容体β鎖可変領域(TCRVβ)のレパトワを８回の解析を試みた。MLPC後，CD8+T細胞中のWT1特異的CTLの割合が高いwellについてレパトワ解析を行った。WT1ペプチドワクチンやMLPCには単一の9merのペプチドを使用したが，３種類のTCRVβの使用が見られた。imatinibとWT1ペプチドワクチン併用療法はCML症例においてWT1特異的CTLを長期間持続させる点で有効であることが確認された。さらに，本研究では，WT1特異的CTLにおいてはoligoclonalなTCRVβが使用されている可能性が示唆された。<br />Antigen specific cytotoxic T lymphocytes (CTLs) play an important role in cancer immunotherapy. To eradicate tumor cells, we administrated WT1 peptide vaccine for a CML patient who was being treated imatinib therapy. The appearance of WT1 specific CTLs in peripheral blood was confirmed by evaluating the frequency of MHC/WT1 tetramer+CD8+ T cells by using mixed lymphocyte peptide culture (MLPC) system. After the cessation of vaccination, WT1 specific CTLs remained at the level of 5-15x10^<-6> in CD8+ T cells, which is lasting thereafter for 6 years. These cells showed cytotoxicity against WT1 peptide with MHC classⅠ restricted. In order to identification of T cell receptor β-chain variable region (TCRVβ) in CML patient received WT1 peptide vaccine, We also investigated the usage of T CVβof WT1 specific CTLs. MLPC cells with high proportion of CD8+ WT1 tetramer+ T cells were assessed for TCRVβ usage by using TCRVβ gene family specific monoclonal antibodies and flow cytometry. Cells from each well cultured by MLPC showed various types of TCRVβ repertoires from well to well. WT1 peptide vaccination for an imatinib pretreated CML patient is effective in terms of longterm generation of WT1 specific CTLs with cytotoxicity against WT1 peptide. The present study suggested that CTLs detected by MLPC possessed oligoclonal features of TCRVβ gene used, but not identical. Taken together, CTLs induced by tumor antigen specific peptide are potent for antitumor immunity.
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| 4.
論文(リポジトリ) |
論文(リポジトリ)
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成田, 美和子 ; 小山, 覚 ; 藤原, 正博 ; 岸, 賢治 ; 高橋, 益広 ; 小池, 正 ; 森山, 美昭 ; 品田, 章二 ; 服部, 晃 ; 柴田, 昭
概要:
As compared with advances in the treatment of acute leukemia, we have made little progress in chronic myelogenous leukem
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ia （CML）. Recentcy we have attempted some new treatment for chronic phass of CML, and confirmed those effectireness. We report the experiences of new therapy in CML.
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