Development of biparatopic bispecific antibody possessing tetravalent scFv-Fc capable of binding to ROBO1 expressed in hepatocellular carcinoma cells

There is no standard structural format of the biparatopic bispecific antibody (bbsAb) which is used against the target molecule because of the diversity of biophysical features of bispecific antibodies (bsAbs). It is therefore essential that the interaction between the antibody and antigen is quantitatively analyzed to design antibodies that possess the desired properties. Here, we generated bsAbs, namely, a tandem scFv-Fc, a diabody-Fc, and an immunofusion-scFv-Fc-scFv, that possessed four scFv arms at different positions and were capable of recognizing the extracellular domains of ROBO1. We examined the interactions between these bsAbs and ROBO1 at the biophysical and cellular levels. Of these, immunofusion-B2212A scFv-Fc-B5209B scFv was stably expressed with the highest relative yield. The kinetic and thermodynamic features of the interactions of each bsAb with soluble ROBO1 (sROBO1) were validated using surface plasmon resonance and isothermal titration calorimetry. In all bsAbs, the immunofusion-scFv-Fc-scFv format showed homogeneous interaction with the antigen with higher affinity compared with that of monospecific antibodies. In conclusion, our study presents constructive information to design druggable bbsAbs in drug applications.