A cell line development vector strategy for improved expression of a trispecific T-cell engager in CHO

Recent advances in trispecific antibody (trisAb) engineering offer great therapeutic potential, but achieving high product yield and quality in cell line development remains a challenge due to complex chain pairing requirements in production cell lines. In this study, three distinct expression vector configurations were evaluated for their ability to support robust, high-level expression of a structurally complex, synapse-gated trisAb T-cell engager (TriMab) in stable Chinese hamster ovary cells. Initial configurations using conventional dual heavy chain (HC) and triple light chain (LC) vectors resulted in poor pool performance characterized by delayed transfection recovery and low titers. By contrast, a redesigned strategy that reversed HC gene order and distributed LCs over separate vectors markedly improved transfection recovery along with product titers and reduced the formation of undesired product variants. Clonal cell lines established with this optimized strategy achieved titers exceeding 2 g/L with correct product quality profiles. Gene copy number and mRNA analyses confirmed that chain order and vector design strongly influenced mRNA levels and thus productivity. These results highlight the critical impact of vector configuration on manufacturability of complex TriMabs, providing a practical framework for the rational design of gene vectors to support next-generation trisAb production.