2003 Fiscal Year Annual Research Report

細胞表面抗原特異的ヒト抗体の網羅的選択・調製と応用

Research Project

Project/Area Number	14704006
Research Institution	Tohoku University
Principal Investigator	津本浩平東北大学, 大学院・工学研究科, 助教授 (90271866)
Keywords	細胞表面抗原 / ファージ提示法 / EGFR / MUCI / 癌免疫療法 / 抗体ライブラリー / 膜蛋白質
Research Abstract	本研究では,申請者が技術基盤を構築してきた,ヒト抗体の安定かつ高効率な人工選択・調製システムを最大限に生かして,細胞表面抗原に対するヒト抗体の網羅的作製を目指すと同時に,ポストゲノム研究で重要な側面を持つ,膜蛋白質を中心とした蛋白質構造・機能評価系への展開を図ることを目的としている.本年度の研究業績は以下の通りである. (1)細胞を使ったヒト抗体の選択:前年度基盤を作った,生細胞を用いたファージ抗体の選択法を,免疫化していない抗体ライブラリーを用いてその至適化を図った.まず,癌抗原と同定されているEGF受容体,ならびに細胞死関連因子TRAIL受容体を形質導入したCHO細胞を準備した.研究代表者らが構築したヒトVH提示ファージライブラリーとヒトVL可溶性分子ライブラリーを用い,抗体VH鎖を濃縮することができた.さらに,ヒトVL提示ライブラリーを用いて同様の選択を行った(2段階選択).癌細胞を用いた選択について,標的抗原を特異的に認識できる分子種の選択法について考察した. (2)自己抗原を用いたヒト抗体の選択:新たにNK細胞阻害性受容体(KIR)特異的ヒト抗体,金表面特異的抗体の選択・調製に成功した.また,BIACORE2000のフロー・リアルタイムモニターリングシステムを用いた,選択・調製法を構築した. (3)各種人工形態蛋白質の構築:癌抗原EGFR特異的抗体(528)を,CD3特異的抗体(OKT3)と融合させた二重特異性抗体Ex3をヒト型化,機能評価等を行い,臨床への具体的展開に大きく前進させたほか,生分解性プラスチック特異的抗体とモデル抗体の融合を図り,その二重特異性と,材料表面への標的分子集積法構築への礎を築いた.

Research Products
(25 results)

All Other

All Publications (25 results)

[Publications] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. 28. 1-8 (2003)
[Publications] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. 278. 5410-5418 (2003)
[Publications] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coil using biotinylated phages"FEBS Letters. 536. 167-172 (2003)
[Publications] Mitsuo Umetsu et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system"The Journal of Biological Chemistry. 278. 8979-8987 (2003)
[Publications] Masahiro Kawahara, et al.: "Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch"Nucleic Acids Research. 31. e32 (2003)
[Publications] Izumi Kumagai, et al.: "Structural consequences of target site-directed functional alteration of an antibody : The case of anti-lysozyme antibody, HyHEL-10"The Journal of Biological Chemistry. 278. 24929-24936 (2003)
[Publications] Mitsunori Shiroishi, et al.: "Human inhibitory receptors ILT2 and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G"Proc.Nati.Acad.Sci.USA. 100. 8856-8861 (2003)
[Publications] Kouhei Tsumoto, et al.: "Immobilized Protein Disulfide Isomerase As an Additive for Refolding of Inclusion Bodies : Application to Antibody Fragments."Protein Engineering. 16. 535-541 (2003)
[Publications] Hiroshi Ueda, et al.: "An optimized homogeneous noncompetitive immunoassay based on the antigen-driven enzymatic complementation"Journal of Immunological Methods. 279. 209-218 (2003)
[Publications] Mitsuhiro Matsumoto, et al.: "On column refolding and characterization of soluble human interleukin-15 receptor a chain produced in Escherichia coil"Protein Expression and Purification. 31. 64-71 (2003)
[Publications] Koji Iida, et al.: "Stable Expression of the Motor Protein Prestin in Chinese Hamster Ovary Cells"JSME International Journal Series C. 46. 1266-1274 (2003)
[Publications] Yoshikazu Tanaka, et al.: "Structural Implications for heavy metal-induced reversible assembly and aggregation of a protein : Crystal structure and metal binding properties of Pyrococcus horikoshii CutA"FEBS Letters. 556. 167-174 (2004)
[Publications] Mitsuo Umetsu, et al.: "Structural characteristics and refolding of in vivo aggregates of archaeon proteins"FEBS Letters. 557. 49-56 (2004)
[Publications] Hiroki Hayashi, et al.: "A highly effective and stable bispecific diabody for cancer immunotherapy : cure of xenografted tumors by bispecific diabody and T-LAK cells."Cancer Immunol Immunother. 53. 495-507 (2004)
[Publications] Masahiro Kawahara, et al.: "AMEGA : antigen-mediated genetically modified cell amplification"Journal of Immunological Methods. 284. 187-194 (2004)
[Publications] Akira Okazaki, et al.: "Fucose Depletion from Human IgGi Oligosaccharide Enhances Binding Enthalpy and Association Rate between IgG1 and Fc□RIIIa"Journal of Molecular Biology. (印刷中). (2004)
[Publications] Yoko, Nagumo, et al.: "Phage-Display Selection of Antibodies to the Left End of CTX3C Using Synthetic Fragments"Journal of Immunological Methods. (印刷中). (2004)
[Publications] Yoshikazu Tanaka, et al.: "Crystal Structure of a Hyperthermophilic CutA from Pyrococcus horikoshii in 3 M Guanidine Hydrochloride : Implication for Guanidine-Side Chain Interactions"Journal of Molecular Biology. (印刷中). (2004)
[Publications] 浅野ら: "組換え型抗体の現状と将来"Bioindustry. 20. 6-14 (2003)
[Publications] 津本, 熊谷: "凝集タンパク質の再生〜凝集抑制と巻き戻し〜"BIOベンチャー. 32-35 (2003)
[Publications] 荒川ら: "タンパク質リフォールデオングの救世主:アルギニン"蛋白質核酸酵素. 48. 2310-2317 (2003)
[Publications] 梅津ら: "凝集蛋白質の効率的再生と分光学的フォールディング研究の接点"生物物理. (印刷中). (2004)
[Publications] 津本, 熊谷(分担執筆): "化学フロンティア(8) コンビナトリアル・バイオエンジニアリング"化学同人. 180 (2003)
[Publications] 熊谷, 津本(分担執筆): "生物物理学ハンドブック"朝倉書店(印刷中). (2004)
[Publications] 津本, 熊谷(分担執筆): "ゲノミクス・プロテオミクスの新展開〜生物情報の解析と応用〜"エヌ・ティー・エス(印刷中). (2004)

2003 Fiscal Year Annual Research Report

細胞表面抗原特異的ヒト抗体の網羅的選択・調製と応用

Principal Investigator

津本 浩平 東北大学, 大学院・工学研究科, 助教授 (90271866)

Research Products

[Publications] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. 28. 1-8 (2003)

[Publications] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. 278. 5410-5418 (2003)

[Publications] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coil using biotinylated phages"FEBS Letters. 536. 167-172 (2003)

[Publications] Mitsuo Umetsu et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system"The Journal of Biological Chemistry. 278. 8979-8987 (2003)

[Publications] Masahiro Kawahara, et al.: "Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch"Nucleic Acids Research. 31. e32 (2003)

[Publications] Izumi Kumagai, et al.: "Structural consequences of target site-directed functional alteration of an antibody : The case of anti-lysozyme antibody, HyHEL-10"The Journal of Biological Chemistry. 278. 24929-24936 (2003)

[Publications] Mitsunori Shiroishi, et al.: "Human inhibitory receptors ILT2 and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G"Proc.Nati.Acad.Sci.USA. 100. 8856-8861 (2003)

[Publications] Kouhei Tsumoto, et al.: "Immobilized Protein Disulfide Isomerase As an Additive for Refolding of Inclusion Bodies : Application to Antibody Fragments."Protein Engineering. 16. 535-541 (2003)

[Publications] Hiroshi Ueda, et al.: "An optimized homogeneous noncompetitive immunoassay based on the antigen-driven enzymatic complementation"Journal of Immunological Methods. 279. 209-218 (2003)

[Publications] Mitsuhiro Matsumoto, et al.: "On column refolding and characterization of soluble human interleukin-15 receptor a chain produced in Escherichia coil"Protein Expression and Purification. 31. 64-71 (2003)

[Publications] Koji Iida, et al.: "Stable Expression of the Motor Protein Prestin in Chinese Hamster Ovary Cells"JSME International Journal Series C. 46. 1266-1274 (2003)

[Publications] Yoshikazu Tanaka, et al.: "Structural Implications for heavy metal-induced reversible assembly and aggregation of a protein : Crystal structure and metal binding properties of Pyrococcus horikoshii CutA"FEBS Letters. 556. 167-174 (2004)

[Publications] Mitsuo Umetsu, et al.: "Structural characteristics and refolding of in vivo aggregates of archaeon proteins"FEBS Letters. 557. 49-56 (2004)

[Publications] Hiroki Hayashi, et al.: "A highly effective and stable bispecific diabody for cancer immunotherapy : cure of xenografted tumors by bispecific diabody and T-LAK cells."Cancer Immunol Immunother. 53. 495-507 (2004)

[Publications] Masahiro Kawahara, et al.: "AMEGA : antigen-mediated genetically modified cell amplification"Journal of Immunological Methods. 284. 187-194 (2004)

[Publications] Akira Okazaki, et al.: "Fucose Depletion from Human IgGi Oligosaccharide Enhances Binding Enthalpy and Association Rate between IgG1 and Fc□RIIIa"Journal of Molecular Biology. (印刷中). (2004)

[Publications] Yoko, Nagumo, et al.: "Phage-Display Selection of Antibodies to the Left End of CTX3C Using Synthetic Fragments"Journal of Immunological Methods. (印刷中). (2004)

[Publications] Yoshikazu Tanaka, et al.: "Crystal Structure of a Hyperthermophilic CutA from Pyrococcus horikoshii in 3 M Guanidine Hydrochloride : Implication for Guanidine-Side Chain Interactions"Journal of Molecular Biology. (印刷中). (2004)

[Publications] 浅野ら: "組換え型抗体の現状と将来"Bioindustry. 20. 6-14 (2003)

[Publications] 津本, 熊谷: "凝集タンパク質の再生 〜凝集抑制と巻き戻し〜"BIOベンチャー. 32-35 (2003)

[Publications] 荒川ら: "タンパク質リフォールデオングの救世主:アルギニン"蛋白質核酸酵素. 48. 2310-2317 (2003)

[Publications] 梅津ら: "凝集蛋白質の効率的再生と分光学的フォールディング研究の接点"生物物理. (印刷中). (2004)

[Publications] 津本, 熊谷(分担執筆): "化学フロンティア(8) コンビナトリアル・バイオエンジニアリング"化学同人. 180 (2003)

[Publications] 熊谷, 津本(分担執筆): "生物物理学ハンドブック"朝倉書店(印刷中). (2004)

[Publications] 津本, 熊谷(分担執筆): "ゲノミクス・プロテオミクスの新展開〜生物情報の解析と応用〜"エヌ・ティー・エス(印刷中). (2004)

津本浩平東北大学, 大学院・工学研究科, 助教授 (90271866)

[Publications] 津本, 熊谷: "凝集タンパク質の再生〜凝集抑制と巻き戻し〜"BIOベンチャー. 32-35 (2003)