Establishment of Identification of TCR antigen and prediction of side effects for TCR gene therapy

• Project/Area Number: 18H02689
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 50020:Tumor diagnostics and therapeutics-related
• Research Institution: University of Toyama
• Principal Investigator: Tatsuihko Ozawa 富山大学, 学術研究部医学系, 准教授 (10432105)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2020: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2018: ¥7,280,000 (Direct Cost: ¥5,600,000、Indirect Cost: ¥1,680,000)
• Keywords: T細胞受容体 / TCR遺伝子療法 / がん免疫療法 / ペプチド/MHC / TCR / レパートリー解析 / 抗原同定

Outline of Final Research Achievements

Adoptive T cell therapy using TCR-modified T cells is a promising next-generation immunotherapy. However, it was not easy to isolate a functional TCR, and to analyze the reactivity and predict the side effects. In this study, we have established a method to obtain TCRs that respond to the original cancer cells from tumor-infiltrating lymphocytes, and technically simple method to generate functional p/MHC that analyze the reactivity of TCRs. These results may contribute to development of adoptive T cell therapy using TCR-modified T cells.

Academic Significance and Societal Importance of the Research Achievements

がんに対する新しい治療法として、T細胞が持っているT細胞受容体(TCR)を使った免疫療法、TCR遺伝子療法の研究が進んでいる。しかしながらTCR遺伝子療法に用いたTCRが類似した配列を持つ自己抗原を認識して重篤な副作用を起こす症例が報告されている。本研究では、TCRの反応性を解析する基盤作りを行い、TCRの反応性を解析する簡便なツールであるペプチド/MHCの容易な作製法を確立した。本研究成果を応用することでTCR遺伝子療法のさらなる発展が期待される。

Research Products

• [Journal Article] Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein. (2021)
  • Author(s): Tani H, Kimura M, Tan L, Yoshida Y, Ozawa T, Kishi H, Fukushi S, Saijo M, Sano K, Suzuki T, Kawasuji H, Ueno A, Miyajima Y, Fukui Y, Sakamaki I, Yamamoto Y, Morinaga Y
  • Journal Title: Virology Journal Volume: 18 Issue: 1 Pages: 16-16
  • DOI: 10.1186/s12985-021-01490-7
  • Peer Reviewed / Open Access
• [Journal Article] Characterization of pseudotyped vesicular stomatitis virus bearing the heartland virus envelope glycoprotein. (2021)
  • Author(s): Kimura M, Egawa K, Ozawa T, Kishi H, Shimojima M, Taniguchi S, Fukushi S, Fujii H, Yamada H, Tan L, Sano K, Katano H, Suzuki T, Morikawa S, Saijo M, Tani H.
  • Journal Title: Virology Volume: 556 Pages: 124-132
  • DOI: 10.1016/j.virol.2020.10.006
  • Peer Reviewed / Open Access
• [Journal Article] Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis. (2021)
  • Author(s): Ozawa T, Kobayashi E, Hamana H, Nakamura T, Lyu F, Hayashi A, Muraguchi A, Kishi H
  • Journal Title: Eur. J. Immunol. Volume: in press Issue: 7 Pages: 1850-1853
  • DOI: 10.1002/eji.202049083
  • Peer Reviewed
• [Journal Article] Relationship between T cell receptor clonotype and PD-1 expression of tumor-infiltrating lymphocytes in colorectal cancer. (2020)
  • Author(s): Sukegawa K, Shitaoka K, Hamana H, Kobayashi E, Miyahara Y, Fujii K, Tsuda K, Saeki S, Nagata T, Ozawa T, Saito S, Fujii T, Muraguchi A, Shiku H, Kishi H.
  • Journal Title: Eur. J. Immunol. Volume: 50 Issue: 10 Pages: 1580-1590
  • DOI: 10.1002/eji.201948399
  • Peer Reviewed
• [Journal Article] Physiologic Target, Molecular Evolution, and Pathogenic Functions of a Monoclonal Anti-Citrullinated Protein Antibody Obtained From a Patient With Rheumatoid Arthritis. (2020)
  • Author(s): Ozawa T, Ouhara K, Tsuda R, Munenaga S, Kurihara H, Kohno H, Hamana H, Kobayashi E, Taki H, Tobe K, Sugiyama E, Muraguchi A, Kishi H.
  • Journal Title: Arthritis Rheumatol. Volume: 72 Issue: 12 Pages: 2040-2049
  • DOI: 10.1002/art.41426
  • Peer Reviewed / Open Access
• [Journal Article] A frequent nonsense mutation in exon 1 across certain HLA-A and -B alleles in leukocytes of patients with acquired aplastic anemia. (2020)
  • Author(s): Mizumaki H, Hosomichi K, Hosokawa K, Yoroidaka T, Imi T, Zaimoku Y, Katagiri T, Nguyen MAT, Tran DC, Elbadry MIY, Chonabayashi K, Yoshida Y, Takamatsu H, Ozawa T, Azuma F, Kishi H, Fujii Y, Ogawa S, Tajima A, Nakao S.
  • Journal Title: Haematologica Volume: － Issue: 6 Pages: 247809-247809
  • DOI: 10.3324/haematol.2020.247809
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Production of a rabbit monoclonal antibody for highly sensitive detection of citrus mosaic virus and related viruses. (2020)
  • Author(s): Miyoshi S, Tokunaga S, Ozawa T, Takeda H, Aono M, Miyoshi T, Kishi H, Muraguchi A, Shimizu S, Nozawa A, Sawasaki T.
  • Journal Title: PloS One Volume: 15 Issue: 4 Pages: e0229196-e0229196
  • DOI: 10.1371/journal.pone.0229196
  • Peer Reviewed / Open Access
• [Journal Article] Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model (2020)
  • Author(s): Hamamoto Yuta、Ouhara Kazuhisa、Munenaga Syuichi、Shoji Mikio、Ozawa Tatsuhiko、Hisatsune Jyunzo、Kado Isamu、Kajiya Mikihito、Matsuda Shinji、Kawai Toshihisa、Mizuno Noriyoshi、Fujita Tsuyoshi、Hirata Shintaro、Tanimoto Kotaro、Nakayama Koji、Kishi Hiroyuki、Sugiyama Eiji、Kurihara Hidemi
  • Journal Title: Arthritis Research & Therapy Volume: 22 Issue: 1 Pages: 249-249
  • DOI: 10.1186/s13075-020-02348-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A novel and simple method to produce large amounts of recombinant soluble peptide/major histocompatibility complex monomers for analysis of antigen-specific human T cell receptors (2019)
  • Author(s): Lyu Fulian、Ozawa Tatsuhiko、Hamana Hiroshi、Kobayashi Eiji、Muraguchi Atsushi、Kishi Hiroyuki
  • Journal Title: New Biotechnology Volume: 49 Pages: 169-177
  • DOI: 10.1016/j.nbt.2018.11.005
  • Peer Reviewed
• [Journal Article] Cutting Edge: B cells expressing cyclic citrullinated peptide-specific antigen receptor are tolerized in normal conditions. (2018)
  • Author(s): Yamada H, Ozawa T, Kishi H, Okada S, Nakashima Y, Muraguchi A and Yoshikai Y.
  • Journal Title: J Immunol Volume: 201 Issue: 12 Pages: 3492-3496
  • DOI: 10.4049/jimmunol.1800826
  • Peer Reviewed
• [Journal Article] Autoantibodies reactive to PEP08 are clinically related with morbidity and severity of interstitial lung disease in connective tissue diseases. (2018)
  • Author(s): Okumura M, Ozawa T, Hamana H, Norimatsu Y, Tsuda R, Kobayashi E, Shinoda K, Taki H, Tobe K, Imura J, Sugiyama E, Kishi H, Muraguchi A.
  • Journal Title: Eur J Immunol. Volume: 48 Issue: 10 Pages: 1717-1727
  • DOI: 10.1002/eji.201747381
  • Peer Reviewed
• [Journal Article] Human monoclonal antibodies against West Nile virus from Japanese encephalitis-vaccinated volunteers. (2018)
  • Author(s): Ozawa T, Masaki H, Takasaki T, Aoyama I, Yumisashi T, Yamanaka A, Konishi E, Ohnuki Y, Muraguchi A, Kishi H.
  • Journal Title: Antiviral Res Volume: 154 Pages: 58-65
  • DOI: 10.1016/j.antiviral.2018.04.011
  • Peer Reviewed
• [Journal Article] Isolation of Antigen-Specific, Antibody-Secreting Cells Using a Chip-Based Immunospot Array (2018)
  • Author(s): Kishi Hiroyuki、Ozawa Tatsuhiko、Hamana Hiroshi、Kobayashi Eiji、Muraguchi Atsushi
  • Journal Title: Methods Mol Biol Volume: 1904 Pages: 147-162
  • DOI: 10.1007/978-1-4939-8958-4_6
  • ISBN: 9781493989577, 9781493989584
• [Presentation] Jurkat細胞を用いたTCRのネオアンチゲン反応性解析 (2020)
  • Author(s): 浜名洋, 宮原慶裕, 下岡清美, 祐川健太, 小林栄治, 小澤龍彦, 村口篤, 藤井努, 珠玖洋, 岸裕幸
  • Organizer: 第24回日本がん免疫学会総会
• [Presentation] ISAAC法を用いたTCR様抗体の迅速作製とその応用 (2020)
  • Author(s): 小澤龍彦, 小林栄治, 浜名洋, 村口篤, 岸裕幸
  • Organizer: 第24回日本がん免疫学会総会
• [Presentation] Development of a novel tumor antigen-specific TCR cloning system using a microarray chip. (2020)
  • Author(s): Kobayashi E, Ozawa T, Hamana H, Muraguchi A, Kishi H.
  • Organizer: 第24回日本がん免疫学会総会
• [Presentation] 複数のエピトープと結合するモノクローナル抗体の認識機構ークラスター状結晶からの構造解析ー (2020)
  • Author(s): 小林淳, 小澤龍彦, 正木秀幸, 加藤龍一
  • Organizer: 日本結晶学会2020年度年会
• [Presentation] 新規マラリアワクチン候補PfRiprにより誘導されるウサギモノクローナル抗体の評価 (2020)
  • Author(s): 徳永聡, 長岡ひかる, 小澤龍彦, 岸裕幸, 村口篤, 坪井敬文, 高島英造, 澤崎達也
  • Organizer: 第43回日本分子生物学会
• [Presentation] The identification of a common loss-function mutation across different HLA class alleles frequently detected in patients with acquired aplastic anemia. (2019)
  • Author(s): Mizumaki H, Hosomichi K, Tanabe M, Yoroidaka T, Imi T, Hosokawa K, Katagiri T, Takamatsu H, Ozawa T, Azuma F, Kishi H, Tajima A, Nakao S.
  • Organizer: 24th Congress of European Hematology Association
  • Int'l Joint Research
• [Presentation] A novel TCR cloning system of peptide-specific T cells using immunospot array assay on a chip(T-ISAAC) technology. (2019)
  • Author(s): Kobayashi E, Ozawa T, Hamana H, Shitaoka K, Muraguchi A, Kishi H.
  • Organizer: International Conference on Lymphocyte Engineering 2019
  • Int'l Joint Research
• [Presentation] エイラクブカを用いた抗Venus VNAR抗体の作製 (2019)
  • Author(s): 善家弘貴，武智昭彦，小澤龍彦，宮川拓也，伊東祐二，澤崎達也，竹田浩之
  • Organizer: 第19回日本蛋白質科学会年会・第71回日本細胞生物学会大会 合同年次大会
• [Presentation] 患者HLA遺伝子導入乳がん細胞株を用いた乳がん患者TIL中の腫瘍反応性TCRの探索 (2019)
  • Author(s): 浜名洋，下岡清美，祐川健太，佐伯しおり，長田任一哉，小林栄治，小澤龍彦，藤井努，村口篤，岸裕幸
  • Organizer: 第23回日本がん免疫学会総会
• [Presentation] 腫瘍浸潤CD8+ T細胞におけるPD-1の発現はTCRレパトアに規定される (2019)
  • Author(s): 祐川健太，佐伯しおり，下岡清美，浜名洋，宮原慶裕，小林栄治，長田任一哉，小澤龍彦，藤井努，珠玖洋，村口篤，岸裕幸
  • Organizer: 第23回日本がん免疫学会総会
• [Presentation] Clonally expanded populations of cytotoxic T cell in TILs and peripheral blood in endometrial cancer patients. (2019)
  • Author(s): 津田桂，浜名洋，中島彰俊，森田恵子，津田さやか，島友子，下岡清美，小林栄治，小澤龍彦，村口篤，岸裕幸
  • Organizer: 第23回日本がん免疫学会総会
• [Presentation] 腫瘍浸潤CD8+ T細胞のTCRとPD-1の発現との関連：大腸癌における考察 (2019)
  • Author(s): 岸裕幸，祐川健太，下岡清美，浜名洋，小林栄治，津田桂，長田任一哉，佐伯しおり，小澤龍彦，齋藤滋，藤井努，村口篤
  • Organizer: 第78回日本癌学会学術総会
• [Presentation] ISAAC法を用いたTCR様抗体の迅速単離法の開発 (2019)
  • Author(s): 小澤龍彦，呂福蓮，村口篤，岸裕幸
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] Cloning of tumor antigen-specific TCRs using immunospot array assay on a chip (T-ISAAC) technology. (2019)
  • Author(s): Kobayashi E, Ozawa T, Hamana H, Shitaoka K, Muraguchi A, Kishi H.
  • Organizer: 第48回日本免疫学会学術集会
• [Presentation] Screening of tumor-reactive TCRs from TILs of breast cancer patients using a patients' HLA-transduced breast cancer cell line. (2019)
  • Author(s): Hamana H, Shitaoka K, Sukegawa K, Saeki S, Nagata T, Kobayashi E, Ozawa T, Fujii T, Muraguchi A, Kishi H.
  • Organizer: 第48回日本免疫学会学術集会
• [Presentation] TCR repertoire analysis of cytotoxic T cells in tumor-infiltrated lymphocytes and peripheral blood lymphocytes in endometrial cancers. (2019)
  • Author(s): Tsuda K, Hamana H, Nakashima A, Tsuda S, Shima T, Shitaoka K, Kobayashi E, Ozawa T, Kishi H.
  • Organizer: 第48回日本免疫学会学術集会
• [Presentation] Common TCRb repertoire was found in both decidual and peripheral CD8+ T cells in normal term pregnancy. (2019)
  • Author(s): Morita K, Kobayashi E, Tsuda S, Shitaoka K, Ozawa T, Hamana H, Saito S, Kishi H.
  • Organizer: 第48回日本免疫学会学術集会
• [Presentation] ヒト、ウサギ、サメモノクローナル抗体の迅速作製と創薬への応用 (2019)
  • Author(s): 小澤龍彦
  • Organizer: フォーラム富山「創薬」第49回研究会
  • Invited
• [Presentation] Human anti-WNV monoclonal antibodies established from JEV-vaccinated volunteers. (2019)
  • Author(s): Masaki H, Ozawa T, Takasaki T, Aoyama I, Yumisashi T, Yamanaka A, Konishi E, Muraguchi A, Kishi H.
  • Organizer: ファーマラボEXPOアカデミックフォーラム
• [Presentation] Isolation of monoclonal anti-CCP antibody from RA patient using ISAAC method and identification of autoantigen of the antibody (2019)
  • Author(s): Ozawa T
  • Organizer: Antibody Engineering & Therapeutics Asia 2019
  • Int'l Joint Research
• [Presentation] Physiological target and molecular evolution of ACPA obtained from RA patients (2018)
  • Author(s): Ozawa T, Tsuda R, Hamana H, Kobayashi E, Taki H, Muraguchi A, Kishi H
  • Organizer: 第47回日本免疫学会学術集会
• [Presentation] The development of antigen detection system using yeast surface display library (2018)
  • Author(s): Ohnuki Y, Ozawa T, Hamana H, Kobayashi E, Lyu F, Muraguchi A, Kishi H
  • Organizer: 第47回日本免疫学会学術集会
• [Presentation] Development of TCR-antigen identification system using “cis-interaction” of TCR and peptide/MHC complex on a T cell (2018)
  • Author(s): Yamaguchi S, Kishi H, Shitaoka K, Hamana H, Kobayashi E, Ozawa T, Muraguchi A
  • Organizer: 第47回日本免疫学会学術集会
• [Presentation] TCR repertoire analysis of peptide-specific T cells using immunospot array assay on a chip (T-ISAAC) technology (2018)
  • Author(s): Kobayashi E, Ozawa T, Hamana H, Shitaoka K, Muraguchi A, Kishi H
  • Organizer: 第47回日本免疫学会学術集会
• [Presentation] A rapid and simple protocol for cDNA cloning of tumor antigen-specific TCR (2018)
  • Author(s): Hamana H, Ozawa T, Kobayashi E, Shitaoka K, Muraguchi A, Kishi H
  • Organizer: 第47回日本免疫学会学術集会
• [Presentation] Loss-of function mutations in HLA-classⅠalleles in acquire aplastic anemia: evidence for the involvement of limited classⅠalleles in the auto-antigen presentation of aplastic anemia (2018)
  • Author(s): Mizumaki H, Hosomichi K, Mikoto T, Yoroidaka T, Imi T, Zaimoku Y, Hosokawa K, Katagiri T, Takamatsu H, Ozawa T, Azuma F, Kishi H, Tajima A, Nakano S
  • Organizer: 60th ASH Annual Meeting and Exposition
  • Int'l Joint Research
• [Presentation] 酵母表面ディスプレイ法を用いたTCRの抗原ペプチド/MHCライブラリーの作製とそのスクリーニング (2018)
  • Author(s): 小澤龍彦, 大貫燿, 浜名洋, 小林栄治, 呂福蓮, 村口篤, 岸裕幸
  • Organizer: 第41回日本分子生物学会年会
• [Presentation] 愛媛県産サメを用いた抗Venus VNARの開発と性状解析 (2018)
  • Author(s): 善家弘貴, 成田公義, 武智昭彦, 小澤龍彦, 岸本聡, 伊東祐二, 澤崎達也, 竹田浩之
  • Organizer: 第41回日本分子生物学会年会
• [Presentation] 子宮体癌患者における末梢血CD8T細胞のTCRレパートリー解析 (2018)
  • Author(s): 津田桂, 岸裕幸, 中島彰俊, 浜名洋, 牛島明美, 津田さやか, 島友子, 下岡清美, 小林栄治, 小澤龍彦, 吉野修, 齋藤滋
  • Organizer: 第33回日本生殖免疫学会総会
• [Presentation] 自己免疫性第XIII因子欠乏症例で生じた抗第XIII因子自己抗体のモノクローン化による機能解析 (2018)
  • Author(s): 惣宇利正善, 尾崎司, 小澤龍彦, 村口篤, 一瀬白帝
  • Organizer: 第91回日本生化学会大会
• [Patent(Industrial Property Rights)] TCR様抗体の製造方法およびその利用 (2019)
  • Inventor(s): 小澤龍彦、岸裕幸、村口篤
  • Industrial Property Rights Holder: 小澤龍彦、岸裕幸、村口篤
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2019-202428
  • Filing Date: 2019