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

Han Kee Ong; Ngan T B Nguyen; Jiawu Bi; Yuansheng Yang

doi:10.1093/abt/tbac025

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

Antib Ther. 2022 Oct 13;5(4):288-300. doi: 10.1093/abt/tbac025. eCollection 2022 Oct.

Authors

Han Kee Ong¹, Ngan T B Nguyen¹, Jiawu Bi¹, Yuansheng Yang¹

Affiliation

¹ Bioprocessing Technology Institute, Agency for Science, Technology and Research (ASTAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.

Abstract

Background: Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format 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) at optimal ratios.

Methods: We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production.

Results: 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 titre and correctly assembled product.

Conclusion: The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production.

Keywords: CHO; bispecific antibody; knob-into-hole; recombinase-mediated-cassette-exchange (RMCE); vector design.