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

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29 Citations (Scopus)


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
Issue number8
Publication statusPublished - 2000


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


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