Efficient construction of a diabody using a refolding system: Anti-carcinoembryonic antigen recombinant antibody fragment

Ryutaro Asano, Toshio Kudo, Yukihiro Nishimura, Koki Makabe, Hiroki Hayashi, Masanori Suzuki, Kouhei Tsumoto, Izumi Kumagai

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fvco); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fvmix). All samples refolded with high efficiency; Fvco, Fvmix, scfv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fvsol) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scfv, and diabody).

Original languageEnglish
Pages (from-to)903-909
Number of pages7
JournalJournal of biochemistry
Volume132
Issue number6
DOIs
Publication statusPublished - 2002 Dec 1

Keywords

  • Adoptive immunotherapy
  • Bispecific diabody
  • CEA
  • Inclusion body

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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