抗P糖蛋白single chain Fv抗体とケモカインの融合蛋白作製と応用研究

• 研究課題/領域番号: 13557053
• 研究種目: 基盤研究(B)
• 配分区分: 補助金
• 応募区分: 展開研究
• 研究分野: 呼吸器内科学
• 研究機関: 徳島大学
• 研究代表者: 曽根 三郎 徳島大学, 医学部, 教授 (40145024)
• 研究分担者: 矢野 聖二 徳島大学, 医学部附属病院, 講師 (30294672) ; 西岡 安彦 徳島大学, 医学部, 講師 (70274199) ; 谷 憲治 徳島大学, 医学部, 助教授 (70207166)
• 研究期間 (年度): 2001 – 2002
• 研究課題ステータス: 完了 (2002年度)
• 配分額: 9,800千円 (直接経費: 9,800千円); 2002年度: 4,800千円 (直接経費: 4,800千円); 2001年度: 5,000千円 (直接経費: 5,000千円)
• キーワード: 多剤耐性 / P糖蛋白質 / MRK16 / single chain antibody fragment / P糖蛋白 / single chan抗体 / ケモカイン / 薬剤耐性 / 抗体治療 / 免疫療法 / MRK-16 / MCP-1

研究概要

我々のグループでは、多剤耐性癌細胞に発現するP糖蛋白質を分子標的とし、抗P糖蛋白抗体MRK16、MRK17による耐性癌細胞障害活性について検討してきた。今回、in vivoでの抗腫瘍活性の増強を目的に、MRK16のsingle chain-antibody fragment(scFv)の作製を試みた。最初に、polymerase chain reaction(PCR)法にて、MRK16のハイブリドーマからH鎖およびL鎖の可変領域をクローニングした。シークエンスにて塩基配列を確認の後、リンカーを用いて結合し発現ベクターを作製した。大腸菌にトランスフォーム後、single chain Fv蛋白を精製した(純度>90%)。
MRK16抗体のscFv(scFvMRK16)のP糖蛋白結合能を検討するため、P糖蛋白陽性の多剤耐性肺小細胞癌株SBC-3/DOXを用いてフローサイトメトリーを施行した。P糖蛋白質に対する結合活性は、MRK16抗体と比較して200〜300倍低いものの、scFvMRK16はSBC-3/DOXに結合するが、P糖蛋白質陰性の親株SBC-3には結合活性を示さないことから、P糖蛋白質特異的に結合していることが確認された。またscFvMRK16の結合活性は、MRK16の添加で阻害されることから、MRK16抗体と同じエピトープを認識していることが推測された。
以上から、scFvMRK16は多剤耐性癌細胞に発現するP糖蛋白質をターゲットにしたin vivoのデリバリーシステムに応用可能と思われる。

研究成果

• [文献書誌] Nishimura et al.: "Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells"Hum Gene Ther. 12. 333-346 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
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  • 説明: 「研究成果報告書概要(和文)」より
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  • 説明: 「研究成果報告書概要(和文)」より
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  • 説明: 「研究成果報告書概要(欧文)」より
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  • 説明: 「研究成果報告書概要(欧文)」より
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  • 説明: 「研究成果報告書概要(欧文)」より
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  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Yamamoto, A., Yano, S., Shiraga, M., Ogawa, H., Goto, H., Miki, T., Zhang, H. and Sone, S.: "A Third-Generation Matrix Metalloproteinase (MMP) Inhibitor (ONO-4817) Combined with Docetaxel Suppresses Progression of Lung Micrometastasis of MMP-Expressing Tumor Cells in Nude Mice"Int J Cancer. 103. 822-828 (2003)
  • 説明: 「研究成果報告書概要(欧文)」より
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  • 説明: 「研究成果報告書概要(欧文)」より
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