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

Abstract

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.

Functional Domain Order of an Anti-EGFR × Anti-CD16 Bispecific Diabody Involving NK Cell Activation

“…We have reported that rearranging the domain order affected the cancer growth inhibitory activity of humanized bsDb targeting EGFR and CD3 [ 16 ]. The highest inhibitory effect was observed for the LH type of hEx3-Dbs, and similar tendencies were confirmed with other bsDbs targeting EGFR and CD3 [ 17 , 18 , 25 ]. CD3 co-localizes with a relatively bulky TCR, and a recent study using cryo-electron microscopy clearly revealed the large, fully assembled structure of CD3 and TCR [ 20 ].…”

“…However, we could not isolate and purify soluble hEx16-scDbs for further investigation by using an E. coli expression system. We then used the Brevibacillus choshinensis expression system, which offers several advantages [ 22 , 23 , 24 ], as demonstrated in our recent preparation of small bispecific antibodies, including hEx3-scDbs [ 25 ]. In the present study, we confirmed the successful preparation of soluble hEx16-scDbs using B. choshinensis , followed by gel filtration chromatography and SDS-PAGE analysis under reducing conditions ( Figure 3 C,D and Figure S3A,B ).…”

“…However, increasing the molecular size of the construct, in this case by including a linker, often leads to difficulties in production using a simple E. coli expression system. We recently reported the utility of the B. choshinensis expression system for the preparation of scDbs [ 25 ]. This nonpathogenic Gram-positive bacterium can directly secrete recombinant proteins into culture media without concerns related to contaminating endotoxins [ 22 , 23 , 24 ].…”

“…We previously constructed pROXb3-hEx3-scDb vectors for the expression of hEx3-scDbs in B. choshinensis [ 25 ]. Both genes for hEx16-scDb-HL and hEx16-scDb-LH were amplified by overlapping polymerase chain reactions (PCRs), and then inserted into the pROXb3 expression vector to construct pROXb3-hEx16-scDb-HL for hEx16-scDb-HL and pROXb3-hEx16-scDb-LH for hEx16-scDb-LH, using restriction enzymes.…”

Bispecific Antibodies and Antibody–Drug Conjugates for Cancer Therapy: Technological Considerations

“…By ligating T-cells with target cells and activating T-cells through CD3, blinatumomab can induce the killing of CD19-expressing B-cells including malignant B-cell leukemia and lymphoma [4]. The order of variable domains is critical for the production of functional BiTE [5], while the length of the inter-scFv linker may either be short (GGGGS) or long ((GGGGS) 3 ) without significantly affecting its tumor-killing activity [6]. Of course, it is unlikely that there exists a single optimal molecular configuration for all tandem scFvs, because the effect of variable domain order on the functionality of scFv differs among antibodies, and close proximity of two scFvs connected by a short linker may interfere with the antigen binding activity of certain antibodies.…”