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Thermostabilization of Fab and its application to medicine

Research Project

Project/Area Number 16K08922
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Applied pharmacology
Research InstitutionSojo University

Principal Investigator

Ohkuri Takatoshi  崇城大学, 薬学部, 教授 (70346807)

Co-Investigator(Kenkyū-buntansha) 安楽 誠  崇城大学, 薬学部, 教授 (60398245)
中村 仁美  崇城大学, 薬学部, 助教 (60510691)
Project Period (FY) 2016-04-01 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Keywords抗体工学 / Fab / 抗体医薬 / 糖鎖エンジニアリング / ジスルフィド結合 / 安定化 / 抗体フラグメント / 抗体医薬品 / タンパク質の安定化 / SS結合 / アダリムマブ
Outline of Final Research Achievements

In recent years, antibody drugs have been actively developed and are highly effective against many diseases. In this study, we produced a recombinant protein of Fab molecule of human monoclonal antibody drug adalimumab and produced a modified Fab whose functionality was improved by introducing an amino acid mutation. As a result of designing a novel intermolecular SS bond introduction mutation for the purpose of improving stability, 12 mutants were successfully constructed. we have successfully demonstrated that a Fab mutant with a novel interchain SS bond (H:V177C-L:Q160C) and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. N-glycosylation site was introduced at H-chain constant region of adalimumab Fab through site-directed mutagenesis. We demonstrated that glycosylated Fab can prevent protein aggregation.

Academic Significance and Societal Importance of the Research Achievements

抗体医薬品の欠点の1つは生産コストが高いという点である。本研究において抗体医薬品アダリムアブのFabについて機能を高めた改変Fabを酵母を用いて作製することに成功した。酵母によって安価に生産できるFabが抗体医薬として応用できれば薬価を抑えることが期待できる。また高機能化によりIgG分子より優れた効果を生み出す可能性もうかがえた。本研究成果は、Fabの抗体医薬品への応用につながる重要な知見をもたらしたであろう。

Report

(5 results)
  • 2019 Annual Research Report   Final Research Report ( PDF )
  • 2018 Research-status Report
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (14 results)

All 2020 2019 2018 2017 2016

All Journal Article (4 results) (of which Peer Reviewed: 4 results,  Open Access: 2 results) Presentation (10 results)

  • [Journal Article] C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond2020

    • Author(s)
      Nakamura H, Anraku M, Oda-Ueda N, Ueda T, Ohkuri T
    • Journal Title

      Biological and Pharmaceutical Bulletin

      Volume: 43 Issue: 3 Pages: 418-423

    • DOI

      10.1248/bpb.b19-00612

    • NAID

      130007804448

    • ISSN
      0918-6158, 1347-5215
    • Year and Date
      2020-03-01
    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] A method to induce hen egg lysozyme-specific humoral immune tolerance in mice by pre-exposition with the protein's oligomers.2019

    • Author(s)
      Ohkuri T, Yuge N, Sato K, Ueda T.
    • Journal Title

      Biochem Biophys Rep.

      Volume: 14 Pages: 100679-100679

    • DOI

      10.1016/j.bbrep.2019.100679

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.2018

    • Author(s)
      Nakamura H, Oda-Ueda N, Ueda T, Ohkuri T.
    • Journal Title

      Biochem Biophys Res Commun.

      Volume: 503 Issue: 2 Pages: 752-756

    • DOI

      10.1016/j.bbrc.2018.06.071

    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Journal Article] A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.2018

    • Author(s)
      Nakamura H, Oda-Ueda N, Ueda T, Ohkuri T.
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 495 Issue: 1 Pages: 7-11

    • DOI

      10.1016/j.bbrc.2017.10.140

    • Related Report
      2017 Research-status Report
    • Peer Reviewed
  • [Presentation] ヒト型Fab定常領域への糖鎖配列の導入と糖鎖が及ぼす影響2019

    • Author(s)
      中村仁美、木吉真人、安楽誠、上田直子、植田正、大栗誉敏
    • Organizer
      第92回日本生化学会大会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 分子間SS結合を有したFabのC末端特異的PEG修飾体の調製2019

    • Author(s)
      中村仁美、上田直子、植田正、大栗誉敏
    • 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
    • Related Report
      2019 Annual Research Report
  • [Presentation] ヒト抗体Fabの定常領域へのアミノ酸変異による新規分子間SS結合の導入2019

    • Author(s)
      吉川萌香、財津佑衣、中村仁美、上田直子、植田正、大栗誉敏
    • Organizer
      2019年度 日本生化学会九州支部例会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 抗体医薬品アダリムマブFabの糖鎖付加変異体の解析2018

    • Author(s)
      中村仁美、木吉真人、上田直子、植田正、大栗誉敏
    • Organizer
      日本薬学会第139年会
    • Related Report
      2018 Research-status Report
  • [Presentation] 糖鎖付加によるアダリムマブFabの凝集性の抑制2018

    • Author(s)
      中村仁美、上田直子、植田正、大栗誉敏
    • Organizer
      第18回日本蛋白質科学会年会
    • Related Report
      2018 Research-status Report
  • [Presentation] アダリムマブFabの定常領域での新規SS結合の導入と安定化2018

    • Author(s)
      中村仁美、上田直子、植田正、大栗誉敏
    • Organizer
      日本薬学会第138年会
    • Related Report
      2017 Research-status Report
  • [Presentation] 抗体医薬アダリムマブFab のC末端ジスルフィド結合 が構造安定性に及ぼす影響2017

    • Author(s)
      中村仁美、大栗誉敏、植田正、上田直子
    • Organizer
      日本薬学会第137年会
    • Place of Presentation
      仙台国際センター(宮城県 仙台市)
    • Year and Date
      2017-03-24
    • Related Report
      2016 Research-status Report
  • [Presentation] アダリムマブFabの新規分子間SS結合導入による熱安定化2017

    • Author(s)
      中村仁美、大栗誉敏、植田正、上田直子
    • Organizer
      第17回日本蛋白質科学会年会 (仙台)
    • Related Report
      2017 Research-status Report
  • [Presentation] アダリムマブFabにおける分子間SS結合の熱安定性への影響と新規SS結合の導入2017

    • Author(s)
      中村仁美、上田直子、植田正、大栗誉敏
    • Organizer
      第34回日本薬学会九州支部大会 (熊本)
    • Related Report
      2017 Research-status Report
  • [Presentation] 酵母Pichia pastorisを用 いたアダリムマブFabの調製2016

    • Author(s)
      大栗誉敏 、中村仁美 、植田正 、上田 直子
    • Organizer
      第33回日本薬学会九州支部大会
    • Place of Presentation
      鹿児島大学郡元キャンパス(鹿児島県 鹿児島市)
    • Year and Date
      2016-12-03
    • Related Report
      2016 Research-status Report

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Published: 2016-04-21   Modified: 2021-02-19  

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