TY - JOUR
T1 - Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion
T2 - The case of the hEx3 diabody
AU - Asano, Ryutaro
AU - Kumagai, Takashi
AU - Nagai, Keisuke
AU - Taki, Shintaro
AU - Shimomura, Ippei
AU - Arai, Kyoko
AU - Ogata, Hiromi
AU - Okada, Mai
AU - Hayasaka, Fumitaka
AU - Sanada, Hideaki
AU - Nakanishi, Takeshi
AU - Karvonen, Teemu
AU - Hayashi, Hiroki
AU - Katayose, Yu
AU - Unno, Michiaki
AU - Kudo, Toshio
AU - Umetsu, Mitsuo
AU - Kumagai, Izumi
N1 - Funding Information:
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (R.A. and I.K.) and by grants from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. Additional support was provided through the Advanced Research for Medical Products Mining Programme of the National Institute of Biomedical Innovation (NIBIO).
PY - 2013/5
Y1 - 2013/5
N2 - 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.
AB - 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.
KW - Bispecific diabody
KW - CD3
KW - Cancer immunotherapy
KW - EGFR
KW - Effective domain order
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U2 - 10.1093/protein/gzt009
DO - 10.1093/protein/gzt009
M3 - Article
C2 - 23468569
AN - SCOPUS:84876564677
VL - 26
SP - 359
EP - 367
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
SN - 1741-0126
IS - 5
ER -