Fig 1: Characterization of trastuzumab-optimal-synergistic-epitope (TOSE) -binding monoclonal antibodies. (a) Schematic workflow of trastuzumab-based synergistic functional screening of TOSE-binding antibodies (4H2, 4C9, 4G6, 5F12 and 5G9). 1st screening: ELISA binding and competitive ELISA with trastuzumab or pertuzumab; 2nd screening: binding to HER2-positive cells; 3rd screening: trastuzumab-based cell proliferation inhibition assay. (b) Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) showed trastuzumab-synergistic bioactivity higher than pertuzumab in the BT-474 cell proliferation inhibition assay. TRA: trastuzumab; PER: pertuzumab. (c) The antibody epitope grouping assay was performed by competitive ELISA. Five monoclonal antibodies (4H2, 4C9, 4G6, 5F12 and 5G9) bound to a unique epitope-TOSE, which was different from both the trastuzumab-binding epitope and pertuzumab-binding epitope. (d) The binding specificity and species cross-reactivity were determined by ELISA. 5G9 bound to HER2 protein, but not to other EGFR family members, including EGFR, HER3 and HER4. (e) The cross-species binding reactivity was determined by ELISA. 5G9 was able to bind cynomolgus HER2 protein but not murine HER2 protein. The BT-474 cell proliferation inhibition assay, antibody epitope grouping assay and the cross-reactivity assay were repeated three times and the results were shown as the mean ± SD (n = 3). Two-way ANOVA, ***P<0.001.