A) Spatial localization of MMAF ADC in NCI N87 tumor xenografts grown in the thighs of mice (representative tumor locations indicated by red arrows in P-MMAF injected mouse). 1 nmole of unlabeled antibody (pertuzumab or trastuzumab) or Cy5 labeled P-MMAF or T-MMAF i.v. injected and whole mouse Cy5 fluorescence imaging 24 hours later. Gut auto-fluorescence indicated by green arrow. Scale bar on far right depicts dynamic range of Cy5 epi-fluorescence efficiency of 2.75×10−5 to 10×10−5. B) Anti-tumor efficacy of MMAF ADC in NCI N87 tumor-bearing mice. Mice i.v. injected on day 0 with 1 nmole P-MMAF or T-MMAF. Tumors measured twice a week and plotted as mean tumor volume ± SEM. Statistical significances calculated using two-way ANOVA with Tukey’s multiple comparisons test. C) Kinetics of ADC tumor drug accumulation in NCI N87 tumors. Mice i.v. injected with 1 nmole T-MMAF on day 0. Tumors harvested days 1, 2 and 3 post injection, drug concentrations quantitated and plotted as mean ± SEM. D) Anti-tumor efficacy of MMAF ADC with IR in NCI N87 tumors. Mice i.v. injected on day 0 with 0.5 nmole T-MMAF. 2.5 Gy given on days 1, 2 and 3. Mouse survival plotted and statistical significances calculated using Log-rank (Mantel-Cox) test. E) Anti-HER2 antibody drug delivery in OE19 tumors. Mice i.v. injected on day 0 with 1 nmole P-MMAF or T-MMAF. Tumors harvested days 1, 2 and 3 post injection, drug concentrations measured and plotted as mean ± SEM. Statistical significances calculated using unpaired t-test. F) Spatial localization of systemically delivered MMAF ADC in OE19 tumor xenografts. 1 nmole of P-MMAF i.v. injected and whole animal Cy5 fluorescence imaged 24 hours later. Scale bar on far right depicts dynamic range of Cy5 epi-fluorescence efficiency of 7×10−5 to 10×10−5. G) Anti-tumor efficacy of MMAF ADC with IR in OE19 tumors. Mice i.v. injected on day 0 with 0.5 nmoles pertuzumab or P-MMAF. IR given 2.5 Gy focally on days 1, 2 and 3. Tumors measured twice a week and plotted as mean tumor volume ± SEM. Statistical significances calculated using two-way ANOVA with Tukey’s multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001.