Construction of a diabody (small recombinant bispecific antibody) using a refolding system

Shin Ichi Takemura, Ryutaro Asano, Kouhei Tsumoto, Shinji Ebara, Naoki Sakurai, Yu Katayose, Hideaki Kodama, Hiroshi Yoshida, Masanori Suzuki, Kohzoh Imai, Seiki Matsuno, Toshio Kudo, Izumi Kumagai

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VII specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. 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 ~98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.

Original languageEnglish
Pages (from-to)583-588
Number of pages6
JournalProtein Engineering
Volume13
Issue number8
Publication statusPublished - 2000 Oct 3

Keywords

  • Bispecific antibody
  • Diabody
  • Diabody-T-LAK cell
  • MUC1
  • Refolding system

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Takemura, S. I., Asano, R., Tsumoto, K., Ebara, S., Sakurai, N., Katayose, Y., Kodama, H., Yoshida, H., Suzuki, M., Imai, K., Matsuno, S., Kudo, T., & Kumagai, I. (2000). Construction of a diabody (small recombinant bispecific antibody) using a refolding system. Protein Engineering, 13(8), 583-588.