A novel bicistronic vector inducing balanced alternative splicing for antibody production.

Efficient and stable production of recombinant monoclonal antibodies requires sufficient expression of light (LC) and heavy (HC) chains within host cells. Conventional bicistronic systems such as internal ribosome entry site (IRES)- or 2 A peptide-based vectors often suffer from imbalanced translation efficiencies or the presence of residual peptide tags that can compromise antibody quality. We developed a novel bicistronic expression platform, designated the cassette exon-associated (CAE) system, which exploits alternative splicing derived from the HEG1 gene to generate two mRNA isoforms from a single promoter. The CAE cassette contains synthetic splice donor and acceptor sequences that enable expression of LC and HC without additional peptide sequences. The optimized construct, pCAE3.31, driven by various promoters-including chicken β-actin, cytomegalovirus, and human elongation factor-1α-efficiently expressed functional antibodies in multiple mammalian cell lines. Compared with IRES-based systems, the CAE vector achieved an 8-fold higher antibody yield. Fab fragments were also produced using the CAE system. The CAE system represents a compact and versatile bicistronic expression strategy that ensures expression of LC and HC, avoids extraneous peptide fusion, and is compatible with major mammalian promoters. This platform facilitates efficient production of recombinant antibodies and Fab fragments, supporting screening and potential therapeutic applications.