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MAbs. 2018 Aug/Sep;10(6):854-863. doi: 10.1080/19420862.2018.1476815. Epub 2018 Jul 9.

Comprehensive study of domain rearrangements of single-chain bispecific antibodies to determine the best combination of configurations and microbial host cells.

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a Department of Biotechnology and Life Science, Graduate School of Engineering , Tokyo University of Agriculture and Technology , Tokyo , Japan.
b R&D Department of ProteinExpress Co., Ltd ., Chiba , Japan.
c Advanced Technology Research Laboratories , Idemitsu Kosan Co., Ltd ., Chiba , Japan.
d Joint Department of Biomedical Engineering , University of North Carolina at Chapel Hill and North Carolina State University , Chapel Hill , NC , USA.


Small bispecific antibodies (bsAbs) are important therapeutic molecules and represent the first bsAb format approved by the United States Food and Drug Administration. Diabody (Db), a small bsAb format, has four possible domain orders; we previously reported the differences in the expression levels and cancer growth inhibition effects upon rearranging the domain order of this format. However, there have been no comprehensive reports on domain rearrangements of bispecific single-chain Db (scDb) and tandem single-chain Fv (taFv), which are widely used bsAb formats. In this study, we designed all possible domain orders for scDb and taFv (each with eight variants) with identical Fv pairs and individually expressed all 16 variants using Escherichia coli, Pichia pastoris, and Brevibacillus choshinensis. Comprehensive investigations showed that the intrinsic functions of the variants were similar to each other, regardless of the expression host system, but expression levels varied depending on the format as well as on the host cell. Among the 16 variants, we found a promising candidate that exhibited high activity and productivity. Furthermore, we determined that B. choshinensis is an attractive expression host because of its secretory production of recombinant proteins.


Brevibacillus choshinensis; CD3; Pichia pastoris; cancer immunotherapy; diabody; effective domain order; epidermal growth factor receptor; host cell; small bispecific antibody; tandem scFv

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