For the evaluation of the biological activity of the constructs, we first determined the binding capability of the synthesized bispecific constructs: the 2:1 HER2×CD3 construct and the 2:1 HER2×HER2 negative control by means of flow cytometry. For this, we used HCC1954 (HER2 + CD3 - ) tumor cells and Jurkat (HER2 -CD3 + ) cells (Fig. ). In this study, HCC1954 cells were incubated with either of the synthesized bispecific constructs and then stained with FITC-labeled anti-IgG Fc antibody. This study revealed that isotype control and FITC-labeled anti-IgG Fc alone did not exhibit any cellular binding in the absence of the bispecific construct, whereas both HER2×CD3 TDBA (10a) and HER2×HER2 trivalent constructs (10b) did bind, as expected. Following this, binding of the constructs to Jurkat cells was analyzed in an analogous manner. As expected, only the sample treated with the 2:1 HER2×CD3 construct prior to staining showed an increase in fluorescence compared with untreated control, whilst the samples incubated with trivalent control (10b), isotype control, and FITClabeled anti-IgG Fc antibodies alone did not exhibit any increase in fluorescence. Both results indicate that the synthesized constructs retained their binding capacity to the targeted cells, even after the chemoenzymatic manipulations and purification steps. Secondly, we tested for the presence of interferon-γ in the culture media to determine whether T cell binding was also accompanied by T cell activation (Fig. ). This was done by ELISA, utilizing supernatants that were collected from the coculture or relevant control cultures 48 h after treatment (T cells were obtained from three healthy blood donors). Briefly, T cell/HCC1954 cocultures (effector:target or E:T ratio of 10:1), HCC1954 monocultures or T cell monocultures were incubated with 5 nM of the bispecific construct 10a, or any of the controls (i.e., untreated cells, trivalent HER2 construct 10b, tras[LC]G4Y, MeTz-rbFabCD3, a 1:1 mixture of tras[LC]G4Y and MeTz-rbFabCD3). As shown in Fig. , interferon-γ secretion was observed only in the presence of 2:1 HER2×CD3 TDBA 10a in the T cell/HCC1954 coculture. Importantly, the absence of interferon-γ in the coculture treated with the 1:1 mixture of separate tras[LC]G4Y and MeTz-rbFabCD3 indicates that both building blocks need to be covalently linked in the same molecular construct in order to elicit the desired effector functions. Additionally, no interferon-γ production was observed in T cell monoculture treated with 2:1 HER2×CD3 construct 10a, which suggests that T cell activation would be primarily confined to the tumor microenvironment in vivo, and as such potentially indicates a reduced overstimulation of the immune system in the absence of tumor cells. Now that we confirmed that the constructs retained their affinity for their intended biological targets, and that the T cells were properly activated to release interferon-γ, the stage was set to study the capability of each construct to induce T cell-mediated cell death of HER2 + HCC1954 tumor cells (Fig. ). For this, monocultures of T cells or HCC1954, and coculture of T cell/HCC1954 cells (E:T ratio of 10:1) were incubated with 5 nM of each construct, and HCC1954 cell viability was measured 48 h post treatment. To our delight, >90% tumor cell death was observed in cocultures treated with the 2:1 HER2×CD3 TDBA 10a, whilst no significant reduction in cell viability was observed with any of the other constructs. The fact that a mixture of the non-linked components did not trigger T cell mediated cell killing underscores that simultaneous binding of T cells and tumor cells by the same molecular construct is required for proper T cell activation against tumor cells. Lastly, the IC50 of 2:1 HER2×CD3 construct 10a was determined using HCC1954 cells and T cells (E:T ratio of 10:1) (Fig. ). Exposure of cocultures to increasing concentrations of our T cell-engaging bispecific antibody construct 10a revealed very low IC50 values of 2.4-12.2 pM. In view of our observation that the tested sample contained 71% of the 2:1 bispecific construct -the other major component, 24%, was identified as the inactive construct tras[LC]G4-TCO (9)-it can safely be concluded that the reported activities are conservative indications of the actual activity of the bispecific construct that was prepared. In comparison to the IC50 values obtained for our earlier described 2:2 HER2×CD3 bispecific antibody constructs (which ranged from 1.3 pM to 26.7 pM), it appears that this new synthetic platform leads to 2:1 bispecific conjugates that are at least as active as the 2:2 bispecifics.