Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

• DOI: 10.4161/mabs.29445
• Publication Date: 2014-06-06T14:05:48Z
• Authors: Ryutaro Asano; Ippei Shimomura; Shota Konno; Akiko Ito; Yosuke Masakari; Ryota Orimo; Shintaro Taki; Kyoko Arai; Hiromi Ogata; Mai Okada; Shozo Furumoto; Masayoshi Onitsuka; Takeshi Omasa; Hiroki Hayashi; Yu Katayose; Michiaki Unno; Toshio Kudo; Mitsuo Umetsu; Izumi Kumagai
• Keywords: Fragment crystallizable region; Bispecific antibody

One approach to creating more beneficial therapeutic antibodies is develop bispecific (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding each target antigen. Although the effects of configuration and antibody-fragment type on function bsAbs have been studied, there only a few detailed studies influence variable fragment domain order. Here, we prepared four types hEx3-scDb-Fc, bsAbs, built from single-chain hEx3-Db (humanized diabody [bsDb] that targets epidermal growth factor receptor CD3), investigate order fusion manner bsDb an Fc format. Higher cytotoxicities were observed hEx3-scDb-Fcs light (VL)–variable heavy (VH) (hEx3-scDb-Fc-LHs) compared VH–VL order, indicating differences in do not affect activity. In addition, flow cytometry suggested higher hEx3-scDb-Fc-LH be attributable structural superiority cross-linking. Interestingly, enhanced degradation resistance prolonged vivo half-life also hEx3-scDb-Fc-LH. its IgG2 variant exhibited intense antitumor effects, suggesting Fc-mediated effector functions are dispensable for effective anti-tumor activities, cause fewer side effects. Our results show merely rearranging can enhance their activity, but half-life, hEx3-scDb-Fc-LHs potent candidates next-generation antibodies.