Adoptive transfer of genetically engineered human cells secreting bispecific T cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs, because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 x anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T cell activation and cytotoxicity against CEA-positive tumor cells.