Abstract
Abstract
Bispecific antibodies targeting two surface receptors have recentl attracted attention in cancer treatment. In this study, we constructed a bispecific antibody against human epidermal growth factor receptor 2 (HER2/ERBB2) and HER3/ERBB3 using the Knobs-into-Holes (KIH) strategy. The expression vectors of anti-HER2 VHH-FcK (Knob) and anti-HER3 scFv-FcK (Hole) genes were transferred into
Agrobacterium tumefaciens
, respectively, and co-infiltrated into
Nicotiana benthamiana
for transient expression. Anti-HER2/HER3 bispecific antibody expression was confirmed by RT-PCR and Western blot. The bispecific antibody was purified by protein A affinity chromatography. The size and purity of the antibody were confirmed by SDS-PAGE and SEC-HPLC analyses. The dual binding activity of anti-HER2/anti-HER3 bispecific antibody was confirmed to bind HER2 and HER3, respectively, by ELISA. In silico structural modeling demonstrated that the KIH Fc configuration favors stable heterodimer formation over homodimeric associations. These results demonstrate that co-infiltration-based transient expression with the KIH strategy can be used in combination to efficiently produce anti-HER2/HER3 bispecific antibody in plant.