Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: The case of the hEx3 diabody

Ryutaro Asano, Takashi Kumagai, Keisuke Nagai, Shintaro Taki, Ippei Shimomura, Kyoko Arai, Hiromi Ogata, Mai Okada, Fumitaka Hayasaka, Hideaki Sanada, Takeshi Nakanishi, Teemu Karvonen, Hiroki Hayashi, Yu Katayose, Michiaki Unno, Toshio Kudo, Mitsuo Umetsu, Izumi Kumagai

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.

Original languageEnglish
Pages (from-to)359-367
Number of pages9
JournalProtein Engineering, Design and Selection
Volume26
Issue number5
DOIs
Publication statusPublished - 2013 May

Keywords

  • Bispecific diabody
  • CD3
  • Cancer immunotherapy
  • EGFR
  • Effective domain order

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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