1999 Fiscal Year Final Research Report Summary
Establishment of the adoptive anticancer immunotherapy for the unresectable cholangiocarcinoma using the bispecific antibody - Construction of bacterial expression system of bispecific antibodies
| Project/Area Number |
09557102
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Digestive surgery
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
SUZUKI Masanori Tohoku University Hospital, Research Associate, 医学部・附属病院, 助手 (70206530)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAGAI Izumi Graduate School of Engineering, Tohoku University, Professor, 大学院・工学研究科, 教授 (10161689)
ENDO Kojin Tohoku University Hospital, Research Associate, 医学部・附属病院, 助手 (70292315)
UNNO Michiaki Graduate School of Medicine, Tohoku University, Research Associate, 大学院・医学系・研究科, 助手 (70282043)
TSUMOTO Kohei Graduate School of Engineering, Tohoku University, Research Associate, 大学院・工学研究科, 助手 (90271866)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Adoptive immunotherapy / Cholangiocarcinoma / Bispecific antibody / MUC 1 / MUSE 11 / SEA / single chain Fv |
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| Research Abstract |
In order to construct the stabel supply system and apply to the immunotherapy for unresectable cholangiocarcinoma, the bacterial expression system of variable regions of anti-MUC-1 IgG, anti-CD3 IgG and anti-CD28 IgG derived from original hybridoma cell line was constructed. Each cDNA encoding corresponding variable region of immunoglobulin was cloned, followed by linking the regions with flexible peptide linker (GlyGlyGlyGlySer)3. The genes encoding single chain Fv fragment were arranged with either VH-linker-VL or VL-linker-VH. The expressed insoluble proteins were solubillized by Guanidine-HCl, refolded, and purified by metal-chelating chematography. Prepared MUSE-11, anti-CD28 and anti-CD3 scFv had same specificity for each original IgG. And a bispecific diabody binding to adenocarcinoma associated antigen MUC1 and to CD3 on Tcells has been focused. Namely, one chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). They second was composed of the VL specific for MUC1 linked to the VH specific for CD3. The two hetero sc Fvs were independently obtained from intracellular insoluble fractions of E. coli, purified and mixed stoichiometrically and refolded by the modified dialysis method. The refolded two hetero scFv has hetero-dimeric structure, with distinct specificity for both target cells. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach about 98% at the effector:target ratio of 10, almost identical to that with anti-MUC1 x anti-CD3 chemically synthesized BsAbs. Bispecific diabody may be good candidates for new adoptive targeted immunotherapy.
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[Publications] Shinoda M, Kudo T, Suzuki M, Katayose Y, Sakurai N, Saeki H, Kodama H, Fukuhara K, Imai K, Hinoda Y, Matsuno S: "Effective adoptive immunotherapy by T-LAK cells retargeted with bacterial superantigen-conjugated antibody to MUCI in xenografted severe combinded"Cancer Research. 58. 2838-2843 (1998)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Sakurai N, Kudo T, Suzuki M, Tsumoto K, Takemura S, Kodama H, Ebara S, Teramae A, Katayose Y, Shinoda M, Kurokawa T, Hinoda Y, Imai K, Matsuno S, Kumagai I: "SEA-scFv as a Bifunctional antibody : Construction of a bacterial expression system and its functional analysis"BBRC. 256(1). 223-230 (1999)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Sakurai N, Kudo T, Suzuki M, Tsumoto K, Takemura S, Kodama H, Ebara S, Teramae A, Katayose Y, Shinoda M, Matsuno S, Kumagai I: "Construction of bacterial expression system of anti-CD28 single-chain antibody"FEBS Letter. (submitted).
Description
「研究成果報告書概要(欧文)」より
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[Publications] Takemura S, Kudo T, Asano R, Tsumoto K, Ebara S, Sakurai N, Katayose Y, Kodama H, Yoshida H, Suzuki M, Imai K, Matsuno S, Kumagai I: "Construction of a diabody (small recombinant bispecific antibody) using an improved refolding system : a case of dabody specific to MUC1 and CD3 for cancer immunotherapy (Protein engineering)"(submitted).
Description
「研究成果報告書概要(欧文)」より
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[Publications] Asano, R, Takemura S, Tsumoto K, Sakurai N, Teramae A, Ebara S, Katayose Y, Masao Shinoda, Suzuki M, Imai K, Matsuno S, Kumagai I: "Functional construction of anti-mucin core protein (MUC1) antibody, MUSE11 variable regions in bacterial expression system : Drastic reduction of binding activity by linking variable domains (Protein engineering)"(submitted).
Description
「研究成果報告書概要(欧文)」より
