Cell-free display technologies for development of next-generation therapeutic antibody fragments

• Project/Area Number: 25289298
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Keio University
• Principal Investigator: DOI NOBUHIDE 慶應義塾大学, 理工学部, 准教授 (50327673)
• Co-Investigator(Kenkyū-buntansha): YANAGAWA HIROSHI 慶應義塾大学, 理工学部, 訪問教授 (40327672)
• Co-Investigator(Renkei-kenkyūsha): MATSUSHIMA KOJI 東京大学, 大学院医学系研究科, 教授 (50222427)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2015: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2014: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2013: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: 蛋白質 / 進化 / バイオテクノロジー / がん / 免疫学

Outline of Final Research Achievements

In this study, we have modified cell-free display technologies previously developed in our laboratory and applied them to efficient and rapid selection of next-generation therapeutic antibody fragments with novel function such as high effector function and bispecificity. We obtained following results. (1) We selected a novel Fc receptor-binding peptide using conventional mRNA display. (2) We improved mRNA display of antibody fragments based on PURE system as a cell-free protein synthesis system and successfully applied it to in vitro selection of a novel humanized scFv against GPCR. (3) We established covalent bicistronic DNA display and applied it to directed evolution of Fab fragments with high thermostability and in vitro selection of bispecific diabodies.

Research Products

• [Journal Article] Endosomal escape efficiency of fusogenic B18 and B55 peptides fused with anti-EGFR single chain Fv as estimated by nuclear translocation. (2016)
  • Author(s): Niikura, K., Horisawa, K., Doi, N.
  • Journal Title: J. Biochem. Volume: 159 Issue: 1 Pages: 123-132
  • DOI: 10.1093/jb/mvv083
  • NAID: 40020725715
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] PURE mRNA display for in vitro selection of single-chain antibodies. (2016)
  • Author(s): Nagumo, Y., Fujiwara, K., Horisawa, K., Yanagawa, H., Doi, N.
  • Journal Title: J. Biochem. Volume: 159 Issue: 5 Pages: 519-526
  • DOI: 10.1093/jb/mvv131
  • NAID: 40020833577
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape. (2015)
  • Author(s): Niikura, K., Horisawa, K., Doi, N.
  • Journal Title: J. Control. Release Volume: 212 Pages: 85-93
  • DOI: 10.1016/j.jconrel.2015.06.020
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display (2014)
  • Author(s): T. Nagata, K. Shirakawa, N. Kobayashi, H. Shiheido, N. Tabata, Y. Sakuma-Yonemura, K. Horisawa, M. Katahira, N. Doi, H. Yanagawa
  • Journal Title: PLOS ONE Volume: 9 Issue: 10 Pages: e109163-e109163
  • DOI: 10.1371/journal.pone.0109163
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 共有結合型DNAディスプレイ法による二重特異性抗体の試験管内進化 (2015)
  • Author(s): 中山真尚，小宮尚子，藤原慶，堀澤健一，土居信英
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートアイランド（兵庫県・神戸市）
  • Year and Date: 2015-12-03
• [Presentation] PURE mRNAディスプレイ法による低分子抗体の試験管内選択 (2015)
  • Author(s): 海野佑樹，南雲優，藤原慶，堀澤健一，柳川弘志，角田慎一，向洋平，堤康央，土居信英
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートアイランド（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] ヒト由来膜融合促進ペプチドによる生体高分子の細胞内送達 (2015)
  • Author(s): 須藤慧，新倉啓介，藤原慶，土居信英
  • Organizer: 第31回日本DDS学会学術集会
  • Place of Presentation: 京王プラザホテル（東京都・新宿区）
  • Year and Date: 2015-07-02
• [Presentation] PURE mRNAディスプレイ法を用いた翻訳停止ペプチド配列の試験管内選択 (2014)
  • Author(s): 南雲優，水原真実子，藤原慶，堀澤健一，柳川弘志，土居信英
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-27
• [Presentation] 膜融合促進ペプチドB18およびB55による生体高分子の細胞内送達 (2014)
  • Author(s): 新倉啓介，藤原慶，堀澤健一，土居信英
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-26
• [Presentation] 共有結合型バイシストロン性DNAディスプレイ法による抗体Fab断片の試験管内進化 (2014)
  • Author(s): 小宮尚子，住田壮，藤原慶，堀澤健一，柳川弘志，土居信英
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-25
• [Presentation] Bindin由来膜融合促進ペプチドを用いた膜透過抗体および膜透過促進抗体の構築 (2014)
  • Author(s): 新倉啓介，堀澤健一，土居信英
  • Organizer: 第30回日本DDS学会学術集会
  • Place of Presentation: 東京
  • Year and Date: 2014-07-30
• [Presentation] PURE mRNA display for in vitro selection of scFv antibodies (2013)
  • Author(s): 南雲優
  • Organizer: 8th Asian Biophysics Association (ABA) Symposium
  • Place of Presentation: Jeju, Korea
• [Presentation] 配偶子認識蛋白質Bindinに存在するB18ペプチドおよびCoreドメインは細胞膜の透過性を向上させる
  • Author(s): 新倉啓介
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸
• [Book] 進化分子工学 ～高速分子進化によるタンパク質・核酸の開発～ (2013)
  • Author(s): 土居信英
  • Total Pages: 12
  • Publisher: NTS出版
• [Patent(Industrial Property Rights)] 融合タンパク質又は複合タンパク質，細胞内送達用担体，部分ペプチド，細胞膜透過促進剤，DNA，及びベクター (2015)
  • Inventor(s): 須藤慧，新倉啓介，土居信英
  • Industrial Property Rights Holder: 学校法人慶應義塾
  • Industrial Property Rights Type: 特許
  • Filing Date: 2015-06-11