Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity
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Ryutaro Asano1,2, Keisuke Nagai1, Koki Makabe3, Kento Takahashi1, Takashi Kumagai1, Hiroko Kawaguchi1, Hiromi Ogata1, Kyoko Arai1, Mitsuo Umetsu1 and Izumi Kumagai1
1Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan
2Present Address: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
3Graduate School of Science and Engineering, Yamagata University, Yonezawa 992-8510, Japan
Ryutaro Asano, email: firstname.lastname@example.org
Izumi Kumagai, email: email@example.com
Keywords: bispecific diabody; cancer immunotherapy; CD3; EGFR; functional structure
Received: February 28, 2017 Accepted: February 10, 2018 Published: February 14, 2018
We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)–variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH–VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo. Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.
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