Establishment of personalized immunotherapy method for virus-infected diseases using lymphocyte chip

2013 Fiscal Year Final Research Report

• Project/Area Number: 23390264
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Infectious disease medicine
• Research Institution: University of Toyama
• Principal Investigator: MURAGUCHI Atsushi 富山大学, 大学院医学薬学研究部(医学), 教授 (20174287)
• Co-Investigator(Kenkyū-buntansha): KISHI Hiroyuki 富山大学, 大学院・医学薬学研究部(医学), 准教授 (60186210) ; OZAWA Tatsuhiko 富山大学, 大学院・医学薬学研究部(医学), 助教 (10432105) ; KITAJIMA Isao 富山大学, 大学院・医学薬学研究部(医学), 教授 (50214797) ; NIKAIDO Toshio 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

Recently, the emerging and re-emerging infectious diseases such as avian flu and AIDS, and other opportunistic or latent infectious diseases are threatening human beings. Immunotherapy via virus-specific monoclonal antibody and cytotoxic T cells is the most likely candidate to treat these fearful infectious diseases. We had developed a novel and innovative method, called "lymphocyte chip", to isolate human antigen-specific antibody-secreting cells from peripheral blood of patients with infectious diseases. In this research, we improved the lymphocyte chip method to clone efficiently and rapidly (within 10 days) antigen specific-antibody or TCR genes from individual lymphocytes of virus-infected patients as well as cancer patients, which may facilitate personalized immunotherapy for infectious diseases as well as cancer.

Research Products

• [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 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
  • DOI: 10.4161/onci.27258
  • Peer Reviewed
• [Journal Article] A novel cloning and expression system yields and validates TCRs from blood lymphocytes of cancer patients 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: Nat Med 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 novel rabbit immunospot array assay on a chip allows for the rapid generation of rabbit monoclonal antibodies with high affinity (2012)
  • Author(s): Ozawa T, Piao X, Kobayashi E, Zhou Y, Sakurai H, Andoh T, Jin A, Kishi H, Muraguchi A
  • Journal Title: PLoS One Volume: 7(12) Pages: e52383
  • DOI: 10.1371/journal.pone.0052383
  • Peer Reviewed
• [Journal Article] Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8^<+> T-Cell Subsets (2012)
  • Author(s): Sun X, Saito M, Sato Y, Chikata T, Naruto T, Ozawa T, Kobayashi E, Kishi H, Muraguchi A, Takiguchi M
  • Journal Title: PLoS One Volume: 7 Pages: e40386
  • DOI: 10.1371/journal.pone.0040386
  • Peer Reviewed
• [Journal Article] The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody (2012)
  • Author(s): Ozawa T ., Horii M., Kobayashi E., Jin A., Kishi H., and Muraguchi A
  • Journal Title: Biochem. Biophys. Res. Commun Volume: 422 Pages: 245-249
  • DOI: 10.1016/j.bbrc.2012.04.134
  • Peer Reviewed
• [Journal Article] Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array (2011)
  • Author(s): Jin A., Ozawa T ., Tajiri K., Obata T ., Kishi H., Muraguchi A
  • Journal Title: Nat. Protoc Volume: 6 Pages: 668-676
  • DOI: 10.1038/nprot.2011.322
  • Peer Reviewed
• [Journal Article] Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus (2011)
  • Author(s): Ozawa T ., Jin A., T ajiri K., T akemoto M., Okuda T ., Shiraki K., Kishi H., Muraguchi A
  • Journal Title: Antiviral Res Volume: 91 Pages: 283-287
  • DOI: 10.1016/j.antiviral.2011.06.012
  • 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, T ajiri 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: 20131211-13
• [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: 20131211-13
• [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: 20131211-13
• [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: 20131211-13
• [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: 20131211-13
• [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: 20131211-13
• [Presentation] Comparison of a-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: 20131211-13
• [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, T ajiri K, Hatakeyama S, Kobayashi E, Ozawa T, Nagai T , Muraguchi A
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-27
• [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, T aki H, T obe K, Kishi H, Muraguchi A
  • Organizer: 15th International Congress of Immunology
  • Place of Presentation: Milan
  • Year and Date: 20130822-27
• [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: 20130822-27
• [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, T ajiri 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: 20130529-0601
• [Presentation] Rabbit ISAAC (rabbit immunospot array assay on a chip) allows for the rapid generation of rabbit monoclonal antibodies with high affinity (2013)
  • Author(s): Piao X, Muraguchi A, Hamana H, Ozawa T, Kobayashi E, Jin A, Kishi H
  • Organizer: Keystone Symposia 'Antibodies as Drugs'
  • Place of Presentation: Vancouver
  • Year and Date: 20130127-0201
• [Presentation] Detection of antigen-stimulated cytokine secretion in human T -cells at single cell levels on a live cell chip (2012)
  • Author(s): 岸 裕幸, 金 艾順, 小林栄治, 小澤龍彦, 浜名 洋, 村口 篤
  • Organizer: 第41回日本免疫学会学術集会
  • Place of Presentation: 神戸
  • Year and Date: 20121205-07
• [Presentation] The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody (2012)
  • Author(s): 小澤龍彦, 小林栄治, 金 艾順, 岸 裕幸, 村口 篤
  • Organizer: 第41回日本免疫学会学術集会
  • Place of Presentation: 神戸
  • Year and Date: 20121205-07
• [Presentation] Cloning of human antigen-specific TCRs can confer the candidates for cancer gene therapy (2012)
  • Author(s): 小林栄治, 岸 裕幸, 浜名 洋, 小澤龍彦, 中河秀俊, 金 艾順, 村口 篤
  • Organizer: 第41回日本免疫学会学術集会
  • Place of Presentation: 神戸
  • Year and Date: 20121205-07
• [Presentation] Comparison of TCR repertoires in EBV -specific T cells detected by three different staining methods (2012)
  • Author(s): 浜名 洋, 小林栄治, 岸 裕幸, 小澤龍彦, 金 艾順, 村口 篤
  • Organizer: 第41回日本免疫学会学術集会
  • Place of Presentation: 神戸
  • Year and Date: 20121205-07
• [Presentation] Rapid generation of super-antibodies using rabbit ISAAC (2012)
  • Author(s): 朴 秀虹, 小澤龍彦, 小林栄治, 岸 裕幸, 村口 篤
  • Organizer: 第41回日本免疫学会学術集会
  • Place of Presentation: 神戸
  • Year and Date: 20121205-07
• [Presentation] Analysis of cellular responses at single cell levels with a hybrid magnetic microwell array chip (2012)
  • Author(s): Kishi H., Obata T ., T akami S., Kazui M., Ogawa A., Ozawa T., and Muraguchi A
  • Organizer: International Joint Symposium on Single-Cell Analysis
  • Place of Presentation: Kyoto
  • Year and Date: 20121127-28
• [Presentation] A novel human TCR efficient cloning system confers candidate for TCR gene therapy within 10 days (2012)
  • Author(s): Kobayashi E., Mizukoshi E., Kishi H., Hamana H., Nagai T ., Ozawa T., Nakagawa H., Jin A., Kaneko S., and Muraguchi A
  • Organizer: 27th Annual Meeting of Society for Immunotherapy of Cancer
  • Place of Presentation: North Bethesda, USA
  • Year and Date: 20121026-28
• [Presentation] A direct T cell receptor cloning system from single human primary T cells confers the candidate for prospective gene therapy (2012)
  • Author(s): Kishi H
  • Organizer: Gordon Research Conference 'Immunochemistry & Immunobiology'
  • Place of Presentation: Les Diablerets, Switzerland
  • Year and Date: 20120610-15
• [Presentation] Generation and characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus : a candidate for antibody therapeutics for a wide range of influenza A viruses (2011)
  • Author(s): Muraguchi A., Ozawa T, Jin A., T ajiri K., Kishi H
  • Organizer: IBC' s 22nd Annual International Conference 'Antibody Engineering'
  • Place of Presentation: San Diego
  • Year and Date: 20111204-08
• [Presentation] 細胞チップを用いた抗原特異的サイトカイン分泌 T 細胞の単一細胞レベルでの検出 (2011)
  • Author(s): 岸 裕幸, 小林栄治, 小澤龍彦, 浜名 洋, 長井輝美, 田尻和人, 村口 篤
  • Organizer: 第40回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20111127-29
• [Presentation] 可溶化 TCR の作製と機能解析 (2011)
  • Author(s): 小澤龍彦, 小林栄治, 岸 裕幸, 村口篤
  • Organizer: 第40回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20111127-29
• [Presentation] 単一細胞解析に基づく迅速な抗原特異的 T 細胞受容体クローニングシステム (2011)
  • Author(s): 小林栄治, 浜名 洋, 長井輝美, 小澤龍彦, 岸 裕幸, 村口 篤
  • Organizer: 第40回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20111127-29
• [Presentation] ISAAC 法を用いた迅速かつハイスループットなウサギモノクローナル抗体作成法の開発 (2011)
  • Author(s): 朴 秀虹, 小澤龍彦, 小林栄治, 岸 裕幸, 村口 篤
  • Organizer: 第40回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20111127-29
• [Presentation] 迅速な TCR cDNA クローニングシステムを用いたEBV 特異的単一 T 細胞の TCRα/β レパトア解析 (2011)
  • Author(s): 浜名 洋, 小林栄治, 長井輝美, 小澤龍彦, 岸 裕幸, 村口 篤
  • Organizer: 第40回日本免疫学会学術集会
  • Place of Presentation: 千葉
  • Year and Date: 20111127-29
• [Presentation] Detection of antigen-stimulated cytokine-secretion in T-cells at single cell levels on a live cell chip (2011)
  • Author(s): Kishi H., Kobayashi E., Ozawa T ., Hamana H., Nagai T ., T ajiri K., Muraguchi A
  • Organizer: 26th Annual Meeting of Society for Immunotherapy of Cancer
  • Place of Presentation: North Bethesda, USA
  • Year and Date: 20111104-06
• [Presentation] The novel method to detect single antibody-secreting B-cells and single cytokine-secreting T -cells using a cell-microarray chip (2011)
  • Author(s): Ozawa T ., Kobayashi E., T ajiri K., Jin A., Kishi H., Muraguchi A
  • Organizer: Cold Spring Harbor Meeting
  • Place of Presentation: New York
  • Year and Date: 20110722-24
• [Presentation] Single T cell analysis system for rapid cloning and functional evaluation of antigen-specific T cell receptors (2011)
  • Author(s): Kobayashi E., Hamana H., Nagai T ., Horii M., Ozawa T ., Kishi H., Muraguchi A
  • Organizer: Cold Spring Harbor Asia Conference
  • Place of Presentation: Suzhou, China
  • Year and Date: 20110524-28
• [Book] Screening of antigen-specific antibody-secreting cells. Single Cell Analysis, Methods in Molecular Biology 853, editors, Lindström S. and AndersonSvahn H (2012)
  • Author(s): Kishi H., Jin A., Ozawa T ., T ajiri K., Obata T ., and Muraguchi A
  • Total Pages: 141-150
  • Publisher: Humana Press
• [Book] シングル細胞チップと抗体医薬への応用.「ナノ融合による先進バイオデバイス」民谷栄一監修 (2011)
  • Author(s): 小澤龍彦,岸 裕幸,村口 篤
  • Total Pages: 50-57
  • Publisher: シーエムシー出版
• [Patent(Industrial Property Rights)] TCR cDNA の増幅方法 (2014)
  • Inventor(s): 浜名洋, 岸 裕幸, 村口 篤, 下岡清美
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Filing Date: 2014-05-30
• [Patent(Industrial Property Rights)] 抗原特異的T細胞受容体の取得方法 (2014)
  • Inventor(s): 小林栄治, 岸 裕幸, 村口 篤
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2014-007576
  • Filing Date: 2014-01-20
• [Patent(Industrial Property Rights)] T細胞受容体のクローニング方法 (2013)
  • Inventor(s): 村口 篤, 岸 裕幸, 小林栄治, 小澤龍彦
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: PCT/JP2014/52950
  • Filing Date: 2013-07-24
  • Overseas
• [Patent(Industrial Property Rights)] T細胞の刺激方法およびその利用 (2013)
  • Inventor(s): 岸 裕幸, 村口 篤, 浜名洋, 小林栄治, 小澤龍彦
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: PCT/JP2013/056076
  • Filing Date: 2013-03-06
  • Overseas
• [Patent(Industrial Property Rights)] T細胞受容体の抗原同定法および同定用レポーター細胞 (2013)
  • Inventor(s): 岸 裕幸, 村口 篤
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2013-023710
  • Filing Date: 2013-02-08
• [Patent(Industrial Property Rights)] 外来遺伝子導入用ベクター及び外来遺伝子が導入されたベクターの製造方法 (2013)
  • Inventor(s): 堀井雅恵, 岸 裕幸, 小林栄治, 小澤龍彦, 村口 篤
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特許5246904
  • Acquisition Date: 2013-04-19
• [Patent(Industrial Property Rights)] T細胞受容体のクローニング方法 (2012)
  • Inventor(s): 村口 篤, 岸 裕幸, 小林栄治, 小澤龍彦
  • Industrial Property Rights Holder: 国立大学法人富山大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2012-16444
  • Filing Date: 2012-07-25