2001 Fiscal Year Annual Research Report

ゲノム情報に基づく新機能ヒト抗体分子の創製と利用

Research Project

Project/Area Number	13450337
Research Institution	Tohoku University
Principal Investigator	熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)
Co-Investigator(Kenkyū-buntansha)	浅野竜太郎東北大学, 加齢医学研究所, 助手 (80323103) 梅津光央東北大学, 大学院・工学研究科, 助手 (70333846) 津本浩平東北大学, 大学院・工学研究科, 講師 (90271866)
Keywords	抗原抗体相互作用 / 蛋白性抗原 / X線結晶構造解析 / 熱力学的解析 / 分子認識 / 変異体 / 非共有結合 / 蛋白質間相互作用
Research Abstract	ヒトゲノム解析により、ヒト抗体遺伝子群はすでに詳細なゲノム解析が終了しており、これらは天然に存在する蛋白質の機能単位の膨大なライブラリーととらえることができる。本研究では、ヒト抗体ゲノム情報に基づき、(1)遺伝子レベルの多様性の創出、(2)抗体の分子認識能による安定な生体外選択、(3)選択された抗体の高効率な調整系を構築することにより、生体内の免疫系を凌駕するような新機能ヒト抗体分子調整系の構築を目標としている。本年度の研究実績は以下のとおりである。(1)遺伝子多様性創出:Error-pronePCRについて、可変領域遺伝子に適切な条件を見出した。また、CDRライブラリー構築のために、Overlap-PCRによる遺伝子構築の技術基盤を確立した。(2)抗体の分子認識能による安定な選択:VHおよびVL鎖のライブラリーについては、ヒト脾臓由来のcDNAライブラリーより作製した。自己抗原としてヒトエリスロポエチン(hEPO)受容体に対して選択を試み、VH鎖つづいて、VL鎖のファージ抗体ライブラリーから、最適なVLを選び出す、という二段階選択により10-8レベルの抗体を得ることができた。選択効率を高めるための、目的クローンの特異的溶出法についても考察し、特異性に基づく選択の技術基盤を構築した。(3)選択された抗体の高効率な調製系の構築と構造解析:(2)で得られたヒト抗体可変領域は大腸菌の高発現系で封入体として得、巻き戻しにより可溶性分子として調製し、精密な結合定数を測定できた。また、分子認識能の詳細な記述を行うため、NMRによる構造解析を目指しているが、今年度は安定同位体標識を行うシステムを確立した。2種類の抗体断片と各種サイトカインなど免疫賦括剤との融合蛋白質の構築を行い、ガン免疫療法に効果的である可能性を示すことができた。

Research Products

(21 results)

All Other

All Publications (21 results)

[Publications] Yoda et al.: "Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow CD and molecular dynamic simulations"Proteins ; Struct. Funct. Genet.. 42. 49-65 (2001)
[Publications] Arai et al.: "Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer"Analytical Biochemistry. 289. 77-81 (2001)
[Publications] Nakamura et al.: "Panning of phage VH library using nitrocellulose membranes-application to selection of human VH library"The Journal of Biochemistry. 129. 209-212 (2001)
[Publications] Kasai et al.: "Fv fragment of an antibody specific for hepatitis C NS3 protease domain : The sequence analysis, bacterial expression and functional characterization"Biochem. Biophys. Res. Commun. 281. 416-424 (2001)
[Publications] Ueno et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"Anticancer research. 22 印刷中. (2002)
[Publications] 津本: "不溶性顆粒からの蛋白質の巻き戻し"蛋白質核酸酵素. 46. 1238-1246 (2001)
[Publications] 津本, 熊谷: "ファージディスプレーを用いた新規バイオ分子の選択と応用"BIOINDUSTRY. 18. 26-33 (2001)
[Publications] Nakamura et al.: "Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate"Analytical Biochemistry. 印刷中. (2002)
[Publications] Shiroishi et al.: "Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction : The case of HyHEL-10 Fv-hen lysozyme complex"The Journal of Biological Chemistry. 276. 23042-23050 (2001)
[Publications] Kawahara et al.: "Replacing factor dependency with that for lysozyme : Affordable culture of IL-6 dependent hybridoma by transfecting artificial cell surface receptor"Biotechnology and Bioengineering. 76. 416-423 (2001)
[Publications] Nakamura et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 印刷中. (2002)
[Publications] Nakamura et al.: "Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera"J. Biosci. Biotechnol.. 印刷中. (2002)
[Publications] Nakamura et al.: "A growth signal with an artificially induced erythropoietion receptor-gp 130 cytoplasmic domain heterodimer"The Journal of Biochemistry. 130. 305-312 (2001)
[Publications] Horii et al.: "Contribution of Thr29 to the Thermodynamic Stability of Goat α-Lactalbumin As Determined by Experimental and Theoretical Approaches"PROTEINS : Struct. Funct. Genet.. 45. 16-29 (2001)
[Publications] Nakamura et al.: "A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages"Biochem. Biophys. Res. Commun.. 289. 252-256 (2001)
[Publications] Kodama et al.: "Mutated SEA D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma (BDC)"Cancer Immunology Immuntherapy. 50. 539-548 (2001)
[Publications] Nakamura et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting : Application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)
[Publications] Takemura et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 for targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 印刷中. (2002)
[Publications] Kumagai, Tsumoto: "Encyclopedia of Life Sciences"Blackwell 印刷中. (2002)
[Publications] 津本, 熊谷: "コンビナトリアルバイオエンジニアリング"化学同人印刷中. (2002)
[Publications] 津本, 熊谷: "新タンパク質科学実験法第3巻構造機能解析"東京化学同人印刷中. (2002)

2001 Fiscal Year Annual Research Report

ゲノム情報に基づく新機能ヒト抗体分子の創製と利用

Principal Investigator

熊谷 泉 東北大学, 大学院・工学研究科, 教授 (10161689)

Research Products

[Publications] Yoda et al.: "Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow CD and molecular dynamic simulations"Proteins ; Struct. Funct. Genet.. 42. 49-65 (2001)

[Publications] Arai et al.: "Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer"Analytical Biochemistry. 289. 77-81 (2001)

[Publications] Nakamura et al.: "Panning of phage VH library using nitrocellulose membranes-application to selection of human VH library"The Journal of Biochemistry. 129. 209-212 (2001)

[Publications] Kasai et al.: "Fv fragment of an antibody specific for hepatitis C NS3 protease domain : The sequence analysis, bacterial expression and functional characterization"Biochem. Biophys. Res. Commun. 281. 416-424 (2001)

[Publications] Ueno et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"Anticancer research. 22 印刷中. (2002)

[Publications] 津本: "不溶性顆粒からの蛋白質の巻き戻し"蛋白質核酸酵素. 46. 1238-1246 (2001)

[Publications] 津本, 熊谷: "ファージディスプレーを用いた新規バイオ分子の選択と応用"BIOINDUSTRY. 18. 26-33 (2001)

[Publications] Nakamura et al.: "Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate"Analytical Biochemistry. 印刷中. (2002)

[Publications] Shiroishi et al.: "Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction : The case of HyHEL-10 Fv-hen lysozyme complex"The Journal of Biological Chemistry. 276. 23042-23050 (2001)

[Publications] Kawahara et al.: "Replacing factor dependency with that for lysozyme : Affordable culture of IL-6 dependent hybridoma by transfecting artificial cell surface receptor"Biotechnology and Bioengineering. 76. 416-423 (2001)

[Publications] Nakamura et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 印刷中. (2002)

[Publications] Nakamura et al.: "Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera"J. Biosci. Biotechnol.. 印刷中. (2002)

[Publications] Nakamura et al.: "A growth signal with an artificially induced erythropoietion receptor-gp 130 cytoplasmic domain heterodimer"The Journal of Biochemistry. 130. 305-312 (2001)

[Publications] Horii et al.: "Contribution of Thr29 to the Thermodynamic Stability of Goat α-Lactalbumin As Determined by Experimental and Theoretical Approaches"PROTEINS : Struct. Funct. Genet.. 45. 16-29 (2001)

[Publications] Nakamura et al.: "A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages"Biochem. Biophys. Res. Commun.. 289. 252-256 (2001)

[Publications] Kodama et al.: "Mutated SEA D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma (BDC)"Cancer Immunology Immuntherapy. 50. 539-548 (2001)

[Publications] Nakamura et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting : Application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)

[Publications] Takemura et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 for targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 印刷中. (2002)

[Publications] Kumagai, Tsumoto: "Encyclopedia of Life Sciences"Blackwell 印刷中. (2002)

[Publications] 津本, 熊谷: "コンビナトリアルバイオエンジニアリング"化学同人 印刷中. (2002)

[Publications] 津本, 熊谷: "新タンパク質科学実験法 第3巻構造機能解析"東京化学同人 印刷中. (2002)

熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)

[Publications] 津本, 熊谷: "コンビナトリアルバイオエンジニアリング"化学同人印刷中. (2002)

[Publications] 津本, 熊谷: "新タンパク質科学実験法第3巻構造機能解析"東京化学同人印刷中. (2002)