抗体薬物複合体の開発基盤：ヒト由来細胞内侵入抗体の迅速創製システムの構築

• Project/Area Number: 26713007
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Partial Multi-year Fund
• Research Field: Medical pharmacy
• Research Institution: 独立行政法人医薬基盤研究所
• Principal Investigator: 向 洋平 独立行政法人医薬基盤研究所, 創薬支援スクリーニングセンター, サブプロジェクトリーダー (60444501)
• Project Period (FY): 2014-04-01 – 2015-03-31
• Project Status: Discontinued (Fiscal Year 2014)
• Budget Amount: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000); Fiscal Year 2014: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000)
• Keywords: 抗体医薬開発 / ファージディスプレイ / 抗体ライブラリ

Outline of Annual Research Achievements

本申請課題は、理論上あらゆる抗原に対するモノクローナル抗体を取得可能な新規ヒトファージ抗体ライブラリを自ら構築し、その中から抗体薬物複合体（Antibody-Drug Conjugate; ADC）開発に有望な、ヒト由来細胞内侵入抗体を迅速単離可能なシステムの構築を、3年の計画で構築しようとするものである。本システムで開発されるヒト由来細胞内侵入抗体は、標的細胞特異的なターゲティングのみならず、その細胞内への薬物デリバリーをも達成可能なツールとなるため、ヒト由来ADCの開発過程を一挙に短縮可能である点で、他を圧倒する競争力を提示できるものと考えられる。
初年度の達成目標は、高品質なヒト合成ファージ抗体ライブラリの構築にあった。近年、この抗体ライブラリのデザインにおいて、ライブラリを構成する抗体の可変領域VHおよびVLの組み合わせ（VH-VLペア）が、得られる抗体のin vitro、in vivoでの安定性に大きく寄与することが判明し、天然に起こり得ないVH-VLペアの存在によって、抗体ライブラリの品質が大きく損なわれる可能性が指摘されている。本観点から我々は、ヒトの体内で安定的に存在可能な4種のVH-VLペアに着目し、抗原結合部位（CDR）をランダム化した130億種類の一本鎖抗体（scFv）からなる、新規の非免疫ヒト型ファージ抗体ライブラリを構築した。本ライブラリからは、試験した15種全ての抗原に対する抗体が取得でき、scFvでありながら、解離定数10-8 Mの高親和性抗体が取得されるなど、高品質なライブラリであることが判明した。現在、より多種類の抗原に対する抗体取得など、更なる有用性を検証中である。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Expenditure Plan for Carryover Budget

26年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Identification and evaluation of metastasis-related proteins, oxysterol binding protein-like 5 and calumenin in lung tumors. (2015)
  • Author(s): Nagano K, Imai S, Zhao X, Yamashita T, Yoshioka Y, Abe Y, Mukai Y, Kamada H, Nakagawa S, Tsutsumi Y, Tsunoda S.
  • Journal Title: Int J Oncol. Volume: in press
  • Peer Reviewed
• [Journal Article] Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2. (2015)
  • Author(s): Inoue M., Tsunoda S. et al.
  • Journal Title: J. Cell Sci. Volume: 128 Pages: 656-669
  • DOI: 10.1242/jcs.149385
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3 (2015)
  • Author(s): Haruhiko Kamada, Shintaro Taki, Kazuya Nagano, Masaki Inoue, Daisuke Ando, Yohei Mukai, Kazuma Higashisaka, Yasuo Yoshioka, Yasuo Tsutsumi, Shin-ichi Tsunoda
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 456 Issue: 4 Pages: 908-912
  • DOI: 10.1016/j.bbrc.2014.12.030
  • Peer Reviewed
• [Journal Article] Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro. (2014)
  • Author(s): Imai S, Yoshioka Y, Morishita Y, Yoshida T, Uji M, Nagano K, Mukai Y, Kamada H, Tsunoda S, Higashisaka K, Tsutsumi Y.
  • Journal Title: Nanoscale Res Lett. Volume: 9(1) Issue: 1 Pages: 651-651
  • DOI: 10.1186/1556-276x-9-651
  • Peer Reviewed
• [Journal Article] Intestinal absorption and biological effects of orally administered amorphous silica particles. (2014)
  • Author(s): Yoshida T., Yoshioka Y., Takahashi H., Misato K., Mori T., Hirai T., Nagano K., Abe Y., Mukai Y., Kamada H., Tsunoda S., Nabeshi H., Yoshikawa T., Higashisaka K., Tsutsumi Y.
  • Journal Title: Nanoscale Res Lett. Volume: 9 Issue: 1 Pages: 532-532
  • DOI: 10.1186/1556-276x-9-532
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers. (2014)
  • Author(s): Kazuya Nagano, Yuka Maeda, So-ichiro Kanasaki, Takanobu Watanabe, Takuya Yamashita, Masaki Inoue, Kazuma Higashisaka, Yasuo Yoshioka, Yasuhiro Abe, Yohei Mukai, Haruhiko Kamada, Yasuo Tsutsumi, Shin-ichi Tsunoda.
  • Journal Title: Journal of Controlled Release Volume: 189 Pages: 72-79
  • DOI: 10.1016/j.jconrel.2014.06.010
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Eph receptor A10 has a potential as a target for a prostate cancer therapy. (2014)
  • Author(s): Kazuya Nagano, Takuya Yamashita, Masaki Inoue, Kazuma Higashisaka, Yasuo Yoshioka, Yasuhiro Abe, Yohei Mukai, Haruhiko Kamada, Yasuo Tsutsumi, Shin-ichi Tsunoda.
  • Journal Title: Biochemical Biophysical Research Communications. Volume: 450 Issue: 1 Pages: 545-549
  • DOI: 10.1016/j.bbrc.2014.06.007
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter (2014)
  • Author(s): Okada, Y., Funahashi, N., Tanaka, T., Nishiyama, Y., Yuan, L., Shirakura, K., Turjman, A. S., Kano, Y., Naruse, H., Suzuki, A., Sakai, M., Zhixia, J., Kitajima, K., Ishimoto, K., Hino, N., Kondoh, M., Mukai, Y., Nakagawa, S., Garcia-Cardena, G., Aird, W. C., Doi, T.
  • Journal Title: Arterioscler Thromb. Vasc. Biol. Volume: 34 Issue: 7 Pages: 1531-1538
  • DOI: 10.1161/atvbaha.114.303818
  • Peer Reviewed
• [Presentation] Structural study on the TNF and TNFR2 complex. (2015)
  • Author(s): Nakamura T., Mukai Y., Yoshioka Y., Tsunoda S., Yamagata Y., Tsutsumi Y.
  • Organizer: Protein Modifications in Pathogenic Dysregulation of Signaling
  • Place of Presentation: Tokyo (Japan)
  • Year and Date: 2015-01-23 – 2015-01-24
• [Presentation] Optimization of bispecific antibody targeting CD3 and EphA10 as a bio-drug candidate against breast cancer. (2014)
  • Author(s): Taki S., Kamada H., Inoue M., Nagano K., Mukai Y., Tsutsumi Y., Tsunoda S.
  • Organizer: The 19th JFCR-ISCC: New Antitumor Agents under Development in the US, Europe and Japan
  • Place of Presentation: Tokyo (Japan)
  • Year and Date: 2014-12-10 – 2014-12-11
• [Presentation] Optimization of an immune method to shorten time for inducing high-affinity antibodies. (2014)
  • Author(s): Mori T., Mukai Y., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Tsunoda S., Tsutsumi Y.
  • Organizer: Federation of Asian Pharmaceutical Associations Congress 2014
  • Place of Presentation: Sabah (Malaysia)
  • Year and Date: 2014-10-09 – 2014-10-12
• [Presentation] A chemical proteoimcs for searching diagnostic markers of Parkinson’s disease. (2014)
  • Author(s): Misato K., Mukai Y., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Fujio Y., Mochizuki H., Tsunoda S., Tsutsumi Y.
  • Organizer: HUPO 13th Annual World Congress
  • Place of Presentation: Madrid (Spain)
  • Year and Date: 2014-10-05 – 2014-10-08
• [Presentation] The search for heart secretory proteins as heart disease biomarkers. (2014)
  • Author(s): Ogura T., Mukai Y., Maeda M., Fujio Y., Sakata Y., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Tsunoda S., Tsutsumi Y.
  • Organizer: HUPO 13th Annual World Congress
  • Place of Presentation: Madrid (Spain)
  • Year and Date: 2014-10-05 – 2014-10-08
• [Presentation] Analysis of membrane protein expression patterns for cells cultured using Cancer-Tissue Originated Spheroids. (2014)
  • Author(s): Kamada H., Taki S., Inoue M., Nagano K., Mukai Y., Yoshioka Y., Higashisaka K., Tsutsumi Y., Tsunoda S.
  • Organizer: HUPO 13th Annual World Congress
  • Place of Presentation: Madrid (Spain)
  • Year and Date: 2014-10-05 – 2014-10-08
• [Presentation] アカデミア創薬の加速を目指した抗体医薬開発NW構想 (2014)
  • Author(s): 向 洋平
  • Organizer: 第27回大阪大学医工情報連携シンポジウム
  • Place of Presentation: 大阪
  • Year and Date: 2014-09-11
  • Invited
• [Presentation] Basic analysis for screening a novel bone-regenerating peptide from the phage peptide library. (2014)
  • Author(s): Nagano T., Mukai Y., Sugamori Y., Kato G., Ohya K., Aoki K., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Tsunoda S., Tsutsumi Y.
  • Organizer: 41th Annual Meeting & Exposition of the Controlled Release Society.
  • Place of Presentation: Chicago (USA)
  • Year and Date: 2014-07-13 – 2014-07-16
• [Presentation] Expression of Annexin A4 regulates cisplatin-susceptibility in malignant mesothelioma. (2014)
  • Author(s): Nagano K., Yamashita T., Inoue M., Higashisaka K., Yoshioka Y., Abe Y., Mukai Y., Kamada H., Tsutsumi Y., Tsunoda S.
  • Organizer: 23rd European Association for Cancer Research (EACR)
  • Place of Presentation: Munich (Germany)
  • Year and Date: 2014-07-05 – 2014-07-08
• [Presentation] Radiolabeling of anti-VEGFR antibody and gamma camera imaging in rats. (2014)
  • Author(s): Kanai Y., Yoshikawa M., Mukai Y., Okada N., Watabe H., Shimosegawa E., Nakagawa S., Hatazawa, J.
  • Organizer: 2014 SNMMI Annual Meeting
  • Place of Presentation: St. Louis (USA)
  • Year and Date: 2014-06-07 – 2014-06-11
• [Presentation] Creation of a novel non-immune phage human antibody library which consists of four stable VH-VL pairs. (2014)
  • Author(s): Mukai Y., Ogura T., Misato K., Nagano T., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Tsunoda S., Tsutsumi Y.
  • Organizer: 2014 AAPS National Biotechnology Conference
  • Place of Presentation: San Diego (USA)
  • Year and Date: 2014-05-19 – 2014-05-21
• [Presentation] アカデミア創薬の加速を目指した抗体医薬開発ネットワーク構想 (2014)
  • Author(s): 向 洋平
  • Organizer: 抗体医薬開発ネットワークキックオフミーティング
  • Place of Presentation: 大阪
  • Year and Date: 2014-04-18
  • Invited
• [Presentation] Structural change of biopharmaceuticals using ion mobility mass spectrometry analysis. (2014)
  • Author(s): Kamada H., Inoue M., Nagano K., Mukai Y, Higashisaka K., Yoshioka Y., Tsutsumi Y., Tsunoda S.
  • Organizer: 5th FIP Pharmaceutical Sciences World Congress (PSWC)
  • Place of Presentation: Melbourne (Australia)
  • Year and Date: 2014-04-13 – 2014-04-16
• [Presentation] Creation of a novel non-immune phage human antibody library which consists of four stable VH-VL pairs. (2014)
  • Author(s): Mukai Y., Ogura T., Misato K., Nagano T., Higashisaka K., Yoshioka Y., Nagano K., Kamada H., Tsunoda S., Tsutsumi Y.
  • Organizer: 5th FIP Pharmaceutical Sciences World Congress (PSWC)
  • Place of Presentation: Melbourne (Australia)
  • Year and Date: 2014-04-13 – 2014-04-16
• [Presentation] Identification of Annexin A4 as cisplatin-susceptibility related protein in malignant mesothelioma. (2014)
  • Author(s): Nagano K., Yamashita T., Inoue M., Higashisaka K., Yoshioka Y., Abe Y., Mukai Y., Kamada H., Tsutsumi Y., Tsunoda S.
  • Organizer: 5th FIP Pharmaceutical Sciences World Congress (PSWC)
  • Place of Presentation: Melbourne (Australia)
  • Year and Date: 2014-04-13 – 2014-04-16
• [Book] Structural basis for the signal transduction by TNF and TNFR (2015)
  • Author(s): Nakamura T, Mukai Y, Tsutsumi Y, Yamagata Y.
  • Publisher: Protein modifications in pathogenic dysregulation of signaling (Springer)
• [Book] アカデミアからの抗体医薬開発に向けて (2014)
  • Author(s): 向 洋平
  • Total Pages: 6
  • Publisher: 生命科学から創薬へのイノベーション（南山堂）