Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-fc bispecific antibodies in CHO cells

Abstract Background Two-armed FabscFv-Fc is one of the most favoured IgG-like asymmetric bispecific antibody (BsAb) formats due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains: one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc). The use of Fab and scFv in the two arms targeting different antigens respectively overcomes the LC mispairing issue, while efficient heterodimeric HC pairing can be obtained through the knob-into-hole (KIH) technology. However, suboptimal expression ratios of the three polypeptides still result in incorrect chain pairing and difficult-to-remove product-related contaminants. Methods To study how the chain ratios affect the FabscFv-Fc production, we designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to a range of various expression ratios of the three polypeptides and expressed them in stably transfected CHO cells via targeted integration. Results Unbalanced expression of HC and scFvFc resulted in incorrect chain association and reduced antibody titer. Expression of HC and scFvFc chains at 1: 1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titer and correctly assembled product. Conclusion The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production. The vector design strategies presented here can also be applied in optimizing the production of other formats of BsAbs.