リンパ球チップを用いたウイルス感染症に対する個の免疫医療の基盤技術の開発

2013 Fiscal Year Annual Research Report

• Project/Area Number: 23390264
• Research Institution: University of Toyama
• Principal Investigator: 村口 篤 富山大学, 大学院医学薬学研究部(医学), 教授 (20174287)
• Co-Investigator(Kenkyū-buntansha): 岸 裕幸 富山大学, 大学院医学薬学研究部(医学), 准教授 (60186210) ; 小澤 龍彦 富山大学, 大学院医学薬学研究部(医学), 助教 (10432105) ; 北島 勲 富山大学, 大学院医学薬学研究部(医学), 教授 (50214797) ; 二階堂 敏雄 富山大学, 大学院医学薬学研究部(医学), 教授 (50180568)
• Project Period (FY): 2011-04-01 – 2014-03-31
• Keywords: 感染症 / リンパ球チップ / 免疫治療 / 抗体遺伝子 / TCR遺伝子

Research Abstract

【1】取得TCRの機能解析法の確立
取得したTCRの機能を解析することを目的として、1）増幅したTCR遺伝子を相同組換え法により発現ベクターに組込み、レトロウイルスを用いてTCR欠損のT細胞株（TG40）にTCRα/TCRβ遺伝子を導入し、TCR/CD3分子を膜に発現させた。TG40には、あらかじめ、ヒト/マウスのCD4あるいはCD8遺伝子を導入した。2）次に、TCR導入TG40細胞を、ペプチドを発現させた抗原提示細胞あるいはMHC/ペプチドで刺激し、活性化マーカーCD69の発現をFACSで測定、あるいはIL-2産生能をELISAで測定することで、TCRの機能を解析した。この解析法が可能になったことで、抗原特異的T細胞の検出から、TCR遺伝子の取得・機能検証までを全体として約10日間で行える画期的なシステムが確立された。
【2】可溶化TCR作製法の確立
TCRは抗体と異なり膜型蛋白であるために、T細胞に発現させる形でしか扱うことができない。それを可溶化型にすることでモノクローナル抗体と同様に病変部位の検出や治療に応用できる可能性がある。そこで、2種類、すなわち、sTCR（V領域とC領域を含む2量体）とscFv-TCR（単鎖可変領域からなる1量体）のTCR遺伝子を構築した。この遺伝子を大腸菌あるいはCHO細胞に発現させ、可溶化TCRを得た。得られたTCRの親和性は抗体に比して100倍以上低いため、TCRをマルチマー化することで数倍の高親和性の可溶化TCRを得ることができた。

Current Status of Research Progress

Reason

25年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

25年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] A novel system for cloning human TCRs: Cutting short the way to TCR-based anticancer therapy. (2014)
  • Author(s): Kobayashi E, Kishi H, Muraguchi A.
  • Journal Title: Oncoimmunology. Volume: 3 Pages: e27258
  • Peer Reviewed
• [Journal Article] Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells. (2014)
  • Author(s): Kobayashi E, Kishi H, Ozawa T, Horii M, Hamana H, Nagai T, Muraguchi A.
  • Journal Title: Biochem Biophys Res Commun. Volume: 444 Pages: 319-324
  • DOI: 10.1016/j.bbrc.2014.01.049
  • Peer Reviewed
• [Journal Article] A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days. (2013)
  • Author(s): Kobayashi E, Mizukoshi E, Kishi H, Ozawa T, Hamana H, Nagai T, Nakagawa H, Jin A, Kaneko S, Muraguchi A.
  • Journal Title: Nature Medicine Volume: 19 Pages: 1542-1546
  • DOI: 10.1038/nm.3358
  • Peer Reviewed
• [Journal Article] Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells. (2013)
  • Author(s): Ohnaga T, Shimada Y, Moriyama M, Kishi H, Obata T, Takata K, Okumura T, Nagata T, Muraguchi A, Tsukada K.
  • Journal Title: Biomed Microdevices Volume: 15 Pages: 611-616
  • DOI: 10.1007/s10544-013-9775-7
  • Peer Reviewed
• [Journal Article] A rapid and high-throughput quantitation assay of the nuclear factor κB activity using fluorescence correlation spectroscopy in the setting of clinical laboratories. (2013)
  • Author(s): Harada K, Mikuni S, Beppu H, Niimi H, Abe S, Hano N, Yamagata K, Kinjo M, Kitajima I.
  • Journal Title: PLoS One Volume: 8 Pages: e75579
  • DOI: 10.1371/journal.pone.0075579
  • Peer Reviewed
• [Journal Article] Distinct reactivity of the commercially available monoclonal antibodies of D-dimer and plasma FDP testing to the molecular variants of fibrin degradation products. (2013)
  • Author(s): Madoiwa S, Kitajima I, Ohmori T, Sakata Y, Mimuro J.
  • Journal Title: Thromb Res. Volume: 132 Pages: 457-464
  • DOI: 10.1016/j.thromres.2013.08.006
  • Peer Reviewed
• [Journal Article] Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells. (2013)
  • Author(s): Teng Z, Yoshida T, Okabe M, Toda A, Higuchi O, Nogami M, Yoneda N, Zhou K, Kyo S, Kiyono T, and Nikaido T.
  • Journal Title: Cell Transplant Volume: 22 Pages: 267-278
  • DOI: 10.3727/096368912X655055
  • Peer Reviewed
• [Journal Article] Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells. (2013)
  • Author(s): Zhou K, Koike C, Yoshida T, Okabe M, Fathy M, Kyo S, Kiyono T, Saito S, and Nikaido T.
  • Journal Title: Cellular Reprogramming Volume: 15 Pages: 55-67
  • DOI: 10.1089/cell.2012.0021
  • Peer Reviewed
• [Journal Article] Effect of Exogenous Oct4 Overexpression on Cardiomyocyte Differentiation of Human Amniotic Mesenchymal Cells. (2013)
  • Author(s): Nagura S, Otaka S, Koike C, Okabe M, Yoshida T, Fathy M, Fukahara K, Yoshimura N, Misaki T, and Nikaido T.
  • Journal Title: Cellular Reprogramming Volume: 15 Pages: 471-480
  • DOI: 10.1089/cell.2013.0002
  • Peer Reviewed
• [Presentation] Cis-interaction of TCR and antigenic peptides with MHC class I molecules on a CD8+ T-cell. (2013)
  • Author(s): Kishi H, Jin A, Hamana H, Tajiri K, Kobayashi E, Ozawa T, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] Rapid generation of rabbit monoclonal antibodies against phosphorylated peptides and G-protein coupled receptor. (2013)
  • Author(s): Ozawa T, Piao X, Kobayashi E, Jin A, Kishi H, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] Cloning and functional analysis of human telomerase reverse transcriptase (hTERT)-specific TCRs from peptide-vaccinated patients. (2013)
  • Author(s): Kobayashi E, Nakagawa H, Hamana H, Kishi H, Ozawa T, Jin A, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] hTEC4 system that enable us to clone TCR cDNA from antigen specific single T cells with in 4 days. (2013)
  • Author(s): Hamana H, Kobayashi E, Kishi H, Ozawa T, Nakagawa H, Jin A, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] Construction of T cell antigen-exploring system. (2013)
  • Author(s): Piao X, Hamana H, Kishi H, Kobayashi E, Ozawa T, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] Functional analysis of anti-cyclic citrullinated protein antibodies derived from rheumatoid arthritis. (2013)
  • Author(s): Tsuda R, Ozawa T, Kobayashi E, Taki H, Kishi H, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] Comparison of α-fetoprotein-specific TCR repertoires from hepatocellular carcinoma patients after peptide vaccine treatment and healthy donors. (2013)
  • Author(s): Nakagawa H, Kobayashi E, Hamana H, Ozawa T, Kishi H, Muraguchi A.
  • Organizer: 第42回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20131211-20131213
• [Presentation] リン酸化ペプチド及びGPCR特異的ウサギモノクローナル抗体の迅速作製法の開発． (2013)
  • Author(s): 小澤龍彦，朴 秀虹，小林栄治，竹田浩之，澤崎達也，周 越，櫻井宏明，安東嗣修，金 艾順，岸 裕幸，村口 篤．
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸
  • Year and Date: 20131203-20131206
• [Presentation] Therapeutic potential of amnion-derived cells. (2013)
  • Author(s): Nikaido T. , Yoshida T., Okabe M., Koike C.
  • Organizer: Cell Symposia : Using Stem Cells to Model and Treat Human Disease
  • Place of Presentation: Los Angeles
  • Year and Date: 20131121-20131123
• [Presentation] がん抗原特異的TCR遺伝子の迅速クローニング法． (2013)
  • Author(s): 小林栄治，水腰英四郎，岸 裕幸，金子周一，村口 篤．
  • Organizer: 第72回日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 20131003-20131005
• [Presentation] リンパ球チップを用いたヒトモノクローナル抗体の迅速作製． (2013)
  • Author(s): 村口 篤，小澤龍彦，岸 裕幸．
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: 横浜
  • Year and Date: 20130911-20130913
  • Invited
• [Presentation] 末梢血リンパ球中の抗原特異的CD8+T細胞およびT細胞受容体の網羅的・迅速解析． (2013)
  • Author(s): 岸 裕幸，小林栄治，水腰英四郎，小澤龍彦，浜名 洋，長井輝美，中河秀俊，金 艾順，金子周一，村口 篤
  • Organizer: 第5回造血器腫瘍免疫療法研究会学術集会
  • Place of Presentation: 名古屋
  • Year and Date: 20130824-20130824
• [Presentation] Cloning of human antigen-specific TCRs can confer the candidates for cancer gene therapy. (2013)
  • Author(s): Kobayashi E, Mizukoshi E, Kishi H, Hamana H, Nagai T, Ozawa T, Nakagawa H, Jin A, Kaneko S, Muraguchi A.
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-20130827
• [Presentation] A monoclonal CCP-antibody derived from RA patients binds to several citrullinated antigens in the human cellular components. (2013)
  • Author(s): Ozawa T, Tuda R, Kobayashi E, Hounoki H, Shinoda K, Taki H, Tobe K, Kishi H, Muraguchi A.
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-20130827
• [Presentation] Swiprosin-1/EFhd2 is involved in the initial reaction of the IgE Fc receptor signal. (2013)
  • Author(s): Mitsuhashi Y, Hachiya M, Aradate T, Yamanoi Y, Kanamori M, Muraguchi A, Mietenz D, Katagiri T.
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-20130827
• [Presentation] The role of Prohibitin (PHB) in the initial reaction of the IgE Fc receptor signal from lipid raft. (2013)
  • Author(s): Ichii T, Aradate T, Mitsuhashi Y, Hachiya M, Tani N, Yamanoi Y, Kanamori M, Muraguchi A, Katagiri T.
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-20130827
• [Presentation] olyporus alveolaris extract (PAE) have the ability to stimulate macrophages against tumor with unique manner. (2013)
  • Author(s): Sasajima Y, Kawamura M, Aradate T, Muraguchi A, Katagiri T.
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-20130827
• [Presentation] Detection of antigen-stimulated cytokine-secretion in human T-cells at single cell levels on a live cell chip. (2013)
  • Author(s): Kishi H, Jin A, Hamana H, Tajiri K, Hatakeyama S, Kobayashi E, Ozawa T, Nagai T, Muraguchi A.
  • Organizer: Measuring Antigen-Specific Immune Responses (MASIR) 2013
  • Place of Presentation: Dubrovnik
  • Year and Date: 20130529-20130601
• [Presentation] Novel haemostatic biomarkers in acute cardioembolic stroke. (2013)
  • Author(s): Kitajima I, Hirano K, Tani M, Tanaka K
  • Organizer: 20th European Congress of Clinical Chemistry and Laboratory Medicine
  • Place of Presentation: Milan
  • Year and Date: 20130519-20130523
• [Presentation] A New rapid, easyand cost-effective method that identifies unknown pathogenic microorganisms within 3 hours from sample collection. (2013)
  • Author(s): Niimi H, Ueno T, Hayashi S, Mori M, Tabata H, Minami H, Saito S, Kitajima I
  • Organizer: 20th European Congress of Clinical Chemistry and Laboratory Medicine
  • Place of Presentation: Milan
  • Year and Date: 20130519-20130523