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Establishment of the adoptive anticancer immunotherapy for the unresectable cholangiocarcinoma using the bispecific antibody - Construction of bacterial expression system of bispecific antibodies

Research Project

Project/Area Number 09557102
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Digestive surgery
Research InstitutionTOHOKU UNIVERSITY

Principal Investigator

SUZUKI Masanori  Tohoku University Hospital, Research Associate, 医学部・附属病院, 助手 (70206530)

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)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥8,300,000 (Direct Cost: ¥8,300,000)
KeywordsAdoptive immunotherapy / Cholangiocarcinoma / Bispecific antibody / MUC 1 / MUSE 11 / SEA / single chain Fv / MUC-1 / scFu / Bispecific抗体 / LAK細胞 / MVC1 / CD_3 / CD_<28>
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.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] 工藤俊雄他: "ムチンコアMUC1抗原ワクチン細胞の作製"Biotherapy. 11. 549-555 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Shinoda M.et al.: "Effective adoptive immunotherapy by T-LAK cells retargeted with bacterial superantigen-conjugated antibody to MUC1 in xenografed severe combined"Cancer Research. 58. 2838-2843 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sakurai N.et al.: "SEA-scFv as a bifunctional antibody : construction of a bacterial expression system and its functional analysis"BBRC. 256. 223-230 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Takemura S.et al.: "Construction of a diabody using an improved refolding system a case of diabody specific to MUC1 and CD3 for cancer immunotherapy"Protein engineering. (in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Asano R.et al.: "Functional construction of anti MUC1 variable regions in bacterial expression system"Protein engineering. (in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary
  • [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
    • Related Report
      1999 Final Research Report Summary

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Published: 1997-04-01   Modified: 2016-04-21  

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