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.

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Materials and Methods Microorganisms, culture media, and reagentsA. limacinum SR21 (ATCC MYA-1381) was cultured in GPY medium containing 30 g/L glucose, 6 g/L Hipolypepton (Nihon Pharmaceutical, Tokyo, Japan) , 2 g/L yeast extract, and 20 g/L artificial sea salt (Sigma-Aldrich, St. Louis, MO, USA) at pH 6.5, 28℃ and 180 rpm for 48 h. Reagents were purchased from Nacalai Tesque (Osaka, Japan) , Sigma-Aldrich, and Tokyo Kasei (Tokyo, Japan) unless otherwise indicated.

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