Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity

// Ryutaro Asano 1, 2 , Keisuke Nagai 1 , Koki Makabe 3 , Kento Takahashi 1 , Takashi Kumagai 1 , Hiroko Kawaguchi 1 , Hiromi Ogata 1 , Kyoko Arai 1 , Mitsuo Umetsu 1 and Izumi Kumagai 1 1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan 2 Present Address: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan 3 Graduate School of Science and Engineering, Yamagata University, Yonezawa 992-8510, Japan Correspondence to: Ryutaro Asano, email: ryutaroa@cc.tuat.ac.jp Izumi Kumagai, email: kmiz@kuma.che.tohoku.ac.jp Keywords: bispecific diabody; cancer immunotherapy; CD3; EGFR; functional structure Received: February 28, 2017      Accepted: February 10, 2018      Published: February 14, 2018 ABSTRACT 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.