2002 年度実績報告書

細胞表面抗原特異的ヒト抗体の網羅的選択・調製と応用

研究課題

研究課題/領域番号	14704006
研究機関	東北大学
研究代表者	津本浩平東北大学, 大学院・工学研究科, 助教授 (90271866)
キーワード	細胞表面抗原 / ファージ提示法 / EGFR / MUC1 / 癌免疫療法 / 後退ライブラリー / 膜蛋白質
研究概要	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癌抗原と同定されているMUC1をクローニング・形質導入したCHO細胞は準備し,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,まず形質導入していないCHO細胞と混合する(Negative Selection).上清を,標的分子を形質導入している細胞に加え,細胞に特異的に結合するファージ粒子を酸溶出により回収,大腸菌に導入する.この操作を3〜6回程度繰り返すことで,各種抗原特異的抗体VH鎖を濃縮することができた.一方,癌抗原としてEGFRを取り上げ,EGFR特異的抗体528をヒト型化し,その親和性の向上を図った.EGFRを発現していない細胞でさらに,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)ところ,数残基のアミノ酸置換で顕著な親和性の上昇を見出した.細胞を用いた選択の礎を作った. (2)各種融合蛋白質の構築癌抗原特異的抗体(T84.66)を,CD3特異的抗体(OKT3)と融合,二重特異性抗体を作製したほか,スーパー抗原変異体あるいは免疫を賦活するサイトカイン(IL21)と融合させ,その機能・物性を評価し,顕著な細胞障害性の創出をみた.

研究成果
(21件)

すべてその他

すべて文献書誌 (21件)

[文献書誌] Michihiro Nakamura, et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay : application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)
[文献書誌] Shin-ichi Takemura, et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunototherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 51. 33-44 (2002)
[文献書誌] Mishihiro Nakamura, et al.: "Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin-Fluorescein Conjugate"Analytical Biochemistry. 304. 231-235 (2002)
[文献書誌] ARUTO 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. 769-776 (2002)
[文献書誌] MASAHIRO KAWAHARA, et al.: "Selection of Highly Productive Mammalian Cells Based on an Inducible Growth Advantage Using an Antibody/Receptor Chimera"JOURNAL OF BIOSCIENCE AND BIOENGINEERLNG. 93. 399-404 (2002)
[文献書誌] Michihiro Nakamura, et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 520. 77-80 (2002)
[文献書誌] Hideki Watanabe, et al.: "Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens"Biochemical and Biophysical Research Communications. 295. 31-36 (2002)
[文献書誌] Shinobu Honzawa, et al.: "Chiral Recognition in the Binding of Helicenediamine to Double Strand DNA : Interactions Between Low Molecular Weight Helical Compounds and a Helical Polymer"Bioorganic & Medicinal Chemistry. 10. 3213-3218 (2002)
[文献書誌] Kouhei Tsumoto, et al.: "Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4:"FEBS Letters. 520. 77-82 (2002)
[文献書誌] Shusaku Tsumoto, et al.: "Analysis of amino-acid sequences by statistical technique"Information Sciences. 145. 205-214 (2002)
[文献書誌] Ryutaro Asano, et al.: "Antitumor activity of Interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli"FEBS Letters. 528. 70-76 (2002)
[文献書誌] Ryutaro Asano, et al.: "Efficient construction of a diabody using a refolding system : anti-carcinoembryonic antigen recombinant antibody fragment"Journal of Biochemistry. 123. 903-909 (2002)
[文献書誌] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. (印刷中). (2003)
[文献書誌] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. (印刷中). (2003)
[文献書誌] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coli using biotinylated phages"FEBS Letters. (印刷中). (2003)
[文献書誌] Mutsuo Umetsu, et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system : Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment"The Journal of Biological Chemistry. (印刷中). (2003)
[文献書誌] Masahiro Kawahara, et al.: "Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch"Nucleic Acids Research. (印刷中). (2003)
[文献書誌] 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. (印刷中). (2003)
[文献書誌] 津本, 熊谷(分担): "コンビナトリアルサイエンスの新展開"シーエムシー出版. 400 (2002)
[文献書誌] 津本, 熊谷(分担): "新タンパク質科学実験法第3巻構造機能解析(上)"東京化学同人. 200 (2003)
[文献書誌] 津本, 熊谷(分担): "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. 200 (2003)

2002 年度 実績報告書

細胞表面抗原特異的ヒト抗体の網羅的選択・調製と応用

研究代表者

津本 浩平 東北大学, 大学院・工学研究科, 助教授 (90271866)

研究成果

[文献書誌] Michihiro Nakamura, et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay : application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)

[文献書誌] Shin-ichi Takemura, et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunototherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 51. 33-44 (2002)

[文献書誌] Mishihiro Nakamura, et al.: "Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin-Fluorescein Conjugate"Analytical Biochemistry. 304. 231-235 (2002)

[文献書誌] ARUTO 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. 769-776 (2002)

[文献書誌] MASAHIRO KAWAHARA, et al.: "Selection of Highly Productive Mammalian Cells Based on an Inducible Growth Advantage Using an Antibody/Receptor Chimera"JOURNAL OF BIOSCIENCE AND BIOENGINEERLNG. 93. 399-404 (2002)

[文献書誌] Michihiro Nakamura, et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 520. 77-80 (2002)

[文献書誌] Hideki Watanabe, et al.: "Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens"Biochemical and Biophysical Research Communications. 295. 31-36 (2002)

[文献書誌] Shinobu Honzawa, et al.: "Chiral Recognition in the Binding of Helicenediamine to Double Strand DNA : Interactions Between Low Molecular Weight Helical Compounds and a Helical Polymer"Bioorganic & Medicinal Chemistry. 10. 3213-3218 (2002)

[文献書誌] Kouhei Tsumoto, et al.: "Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4:"FEBS Letters. 520. 77-82 (2002)

[文献書誌] Shusaku Tsumoto, et al.: "Analysis of amino-acid sequences by statistical technique"Information Sciences. 145. 205-214 (2002)

[文献書誌] Ryutaro Asano, et al.: "Antitumor activity of Interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli"FEBS Letters. 528. 70-76 (2002)

[文献書誌] Ryutaro Asano, et al.: "Efficient construction of a diabody using a refolding system : anti-carcinoembryonic antigen recombinant antibody fragment"Journal of Biochemistry. 123. 903-909 (2002)

[文献書誌] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. (印刷中). (2003)

[文献書誌] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. (印刷中). (2003)

[文献書誌] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coli using biotinylated phages"FEBS Letters. (印刷中). (2003)

[文献書誌] Mutsuo Umetsu, et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system : Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment"The Journal of Biological Chemistry. (印刷中). (2003)

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

[文献書誌] 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. (印刷中). (2003)

[文献書誌] 津本, 熊谷(分担): "コンビナトリアルサイエンスの新展開"シーエムシー出版. 400 (2002)

[文献書誌] 津本, 熊谷(分担): "新タンパク質科学実験法 第3巻構造機能解析(上)"東京化学同人. 200 (2003)

[文献書誌] 津本, 熊谷(分担): "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. 200 (2003)

2002 年度実績報告書

津本浩平東北大学, 大学院・工学研究科, 助教授 (90271866)

[文献書誌] 津本, 熊谷(分担): "新タンパク質科学実験法第3巻構造機能解析(上)"東京化学同人. 200 (2003)