Optimizing co-expression strategies for the simultaneous display and secretion of monoclonal antibodies in CHO cells.

Mammalian cell-based dual display and secretion (dualDS) systems enable both surface display of antibodies for binding analysis and simultaneous secretion for functional testing within the same cells. While powerful, their efficiency depends heavily on how light (LC) and heavy chains are co-expressed. In this study, we compared three vector designs in Chinese Hamster Ovary cells: (1) multiple promoters (MP), (2) internal ribosomal entry sites (IRES), and (3) a minimal furin cleavage sequence linked to the 2A peptide (Fm-2A). Vectors were stably integrated into a genomic landing pad using recombinase-mediated cassette exchange. Our results show MP constructs produced heterogeneous expression due to integration errors and transcriptional variability. IRES constructs yielded more homogenous expression but suffered from low secretion due to translational inefficiency. In contrast, the Fm-2A design achieved most uniform antibody display and highest secretion, though residual 2A fragments indicated incomplete furin cleavage. Sequence engineering of Fm-2A improved cleavage efficiency and enhanced antibody quality. These findings underscore the importance of vector design in optimizing dualDS systems and provide a framework for improving library screening, engineering, and other mammalian cell-based applications for therapeutic development.