Novel CAE system boosts antibody production 8-fold by balancing light and heavy chain expression

Monoclonal antibodies (mAbs) are crucial biotherapeutics, but their efficient production requires precise, balanced expression of light (LC) and heavy (HC) chains. Traditional bicistronic systems, like those employing internal ribosome entry sites (IRES) or 2A peptides, often lead to imbalanced translation efficiencies between LC and HC. This imbalance can reduce functional antibody yield and quality. Furthermore, 2A peptide systems leave residual tags, which can affect antibody immunogenicity or pharmacokinetics. Addressing these limitations is critical for advancing recombinant antibody manufacturing and ensuring high-quality therapeutic products.

Researchers developed the cassette exon-associated (CAE) system, a novel bicistronic expression platform designed for balanced antibody chain production. They engineered a CAE cassette with synthetic splice donor and acceptor sequences, leveraging alternative splicing from the HEG1 gene to produce distinct light (LC) and heavy (HC) chain mRNA isoforms from a single promoter. The optimized construct, pCAE3.31, was tested with various promoters, including chicken β-actin, cytomegalovirus, and human elongation factor-1α, in multiple mammalian cell lines. Its performance was directly compared against conventional IRES-based bicistronic systems to assess antibody yield and functionality, as well as its ability to produce Fab fragments.

The novel CAE system successfully demonstrated efficient expression of functional antibodies across multiple mammalian cell lines, utilizing various common promoters like chicken β-actin, cytomegalovirus, and human elongation factor-1α. A critical advantage observed was the system's ability to ensure balanced expression of both light (LC) and heavy (HC) chains, circumventing the issues of imbalanced translation seen with other methods. Importantly, the CAE system achieved this without incorporating additional peptide sequences, thus avoiding the residual tags that can compromise antibody quality in 2A peptide-based systems. This streamlined approach contributes to a cleaner and potentially more efficacious final product. The versatility of the CAE system was further confirmed by its successful application in producing functional Fab fragments, highlighting its broad utility for different antibody formats. These findings underscore the CAE system as a robust and compact platform for high-yield, high-quality recombinant antibody production. > Compared with conventional IRES-based bicistronic systems, the optimized pCAE3.31 vector significantly boosted antibody production, achieving an impressive 8-fold higher antibody yield.