Enhanced anti-tumor effect of cell-internalizing mAbs. (A-D) Cytotoxicity of scFv-PSIF and IgG-NCS against MS1 cells. MS1 cells were incubated with serially diluted mAb-drug conjugates for 24 hours. Cell viability was then measured using a WST-8 assay. Closed square, internalizing mAbs; open circle, low-internalizing mAbs; open triangle, negative controls. (A) anti-Robo4[scFv]-PSIFs, (B) anti-Robo4[IgG]-NCSes, (C) anti-VEGFR2[scFv]-PSIFs, (D) anti-VEGFR2[IgG]-NCSes. Anti-His[scFv]-PSIF and anti-FLAG[IgG]-NCS were used as negative controls. Values are shown as means ± SD. (E-H) Antitumor effects of scFv-PSIFs or IgG-NCSes. B16BL6 cells were inoculated intracutaneously into C57BL6 mice on day 0. On days 3, 5, 7, 9, and 11, mAb-drug conjugates were intravenously administered (arrow heads). Tumor volume was calculated using the following formula: tumor volume (mm³) = {major axis of tumor (mm)} × {minor axis of tumor (mm)}² × 0.4. Closed square, internalizing mAbs; open circle, low-internalizing mAbs; open triangle, negative controls (anti-His[scFv]-PSIF or anti-FLAG[IgG]-NCS); open diamond, PBS. (E) Anti-Robo4[scFv]-PSIFs, (F) anti-Robo4[IgG]-NCSes, (G) anti-VEGFR2[scFv]-PSIFs, (H) anti-VEGFR2[IgG]-NCSes. Values are shown as means ± SEM. **P < 0.01; internalizing mAbs versus low-internalizing mAbs by Bonferroni post hoc analysis with two-way ANOVA (n = 6). (I-L) Change in body weight during therapy experiment. Closed square, internalizing mAbs; open circle, low-internalizing mAbs; open triangle, negative controls (anti-His[scFv]-PSIF or anti-FLAG[IgG]-NCS); open diamond, PBS. (I) anti-Robo4[scFv]-PSIFs, (J) anti-Robo4[IgG]-NCSes, (K) anti-VEGFR2[scFv]-PSIFs, (L) anti-VEGFR2[IgG]-NCSes. Values are shown as means ± SEM. **P < 0.01; internalizing mAbs versus PBS by Bonferroni post hoc analysis with two-way ANOVA (n = 6).