Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats

Abstract

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR⁺ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR⁺ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.

Keywords

bispecific antibody / single-chain tandem single-chain variable fragments / diabody / transferrin receptor / CD3

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