Fig. 3: In vivo efficacy of 1F7 and 4B3 mAb in an intravenous metastatic model of PC-3-luciferase cells.

a Schematic of the experimental design. NRG mice received intraperitoneal injections of 1F7, 4B3 or IgG control mAbs (8 mg/kg) or PBS vehicle controls at 2 days and 4 h prior to and 2 days after tail vein injection with PC-3-luciferase cells. In one experimental setup (Protocol 1) mice continued to receive treatments thrice weekly via intraperitoneal injection until endpoint. For Protocol 2, mice received no further injections of antibody after the initial three doses. Tumor burden was monitored using weekly intravital bioluminescence imaging. Representative bioluminescence images at 8 and 9 weeks and quantification of total luminescence flux (pixel/s) from week 5 to 8 after PC-3 cell engraftment (b) and Kaplan–Meier survival analysis (c) of mice treated according to Protocol 1. The box and whisker plot shows the log-scaled median, interquartile range ± maximum/minimum values of the total bioluminescence flux (pixels/s). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between treatment groups, over time and treatments × time. Kaplan–Meier survival curves were analyzed using log-rank test to compare all curves and log-rank (Mantel–Cox) test for pairwise comparisons (n = 7–8 mice per group as indicated), *p < 0.05 and **p < 0.01. Bioluminescence images at 9 and 10 weeks after injection of tumor cells and quantification of total bioluminescence flux (pixel/s) from week 6 to 11 (d) and Kaplan–Meier survival analysis (e) of mice treated according to Protocol 2. The box and whisker plot shows the log-scaled median, interquartile range ± maximum/minimum values of the total bioluminescence flux (pixels/second). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between treatment groups, over time and treatments × time. Kaplan–Meier survival curves were analyzed by log-rank test to compare all curves and log-rank (Mantel–Cox) test for pairwise comparisons (n = 7–8 mice per group as indicated), **p < 0.01 and ***p < 0.001. f Western blot analysis of EphA2 receptor activation and AKT inhibition in HUVEC cells after a 20-min stimulation with ephrin-A1-Fc (efnA1-Fc), clustered ephrin-A1-Fc (cl-efnA1-Fc), 1F7 and 4B3 alone and in combination as indicated. 1F7 blocks EphA2 receptor activation by ephrin-A1-Fc. β-actin is the loading control. g Vascular permeability was measured by Miles Assay. Two representative images from the flanks of two mice showing dye effusion in response to 1F7, 4B3 and control IgG mAbs and PBS. Bar graph shows the relative amount of Evans Blue dye extravasation quantified spectroscopically after formamide extraction from tissue biopsy cores. Mean ± SE (n = 8). *p < 0.05, ***p < 0.001, ns. not significant (Repeated measure, one-way ANOVA with post-hoc Tukey’s test for multiple pairwise comparisons).