Fig. 9: Prot-FOLR1-TCB is efficacious in vivo while there is no hint for tumor-leakage of activated Prot-FOLR1-TCB.

a Tumor growth inhibition curves of breast PDX BC004 model in humanized mice. Humanized mice (n = 9 per group) were weekly i.v. injected with equimolar doses of Prot-FOLR1-TCBs (4 mg/kg) containing different cleavage sites, FOLR1-TCB (3.6 mg/kg) or vehicle. Each dot represents the mean tumor volume ± SEM. Efficacy was evaluated by measuring the reduction of the mean tumor volume at day 62 relative to vehicle control. Statistical analysis was done using one-way ANOVA with Tukey−Kramer correction. No significant efficacy was observed for the Prot-FOLR1-TCB with the noncleavable linker comparing to vehicle control. Significant tumor growth inhibition was induced by the FOLR1-TCB (****p < 0.0001), the Prot-FOLR1-TCB with the matB site (****p < 0.0001) and the Prot-FOLR1-TCB with matC site (*p = 0.034) all compared to vehicle group. The in vivo efficacy study has been conducted once (n = 9 mice per group; n = 8 mice for matB group). b Quantification of human CD3-positive T cells for the different treatment groups of efficacy study using breast PDX in humanized mice. All data points shown in bar chart. Each data point represents the value for one mouse. 95% confidence interval is shown for each group. Two-tailed, unpaired t test was used to calculate statistics. Significantly more huCD3 T cells per mm³ were found for animals treated with FOLR1-TCB (***p = 0.0009) and Prot-FOLR1-TCBs (matC *p = 0.0284 and matB site ***p = 0.0009) than for vehicle group. However also the Prot-FOLR1-TCB with a noncleavable linker had significant more huCD3 T cells per area compared to vehicle (*p = 0.0481). Representative images are shown in Supplementary Fig. 9. c, d Bioavailability of active Prot-FOLR1-TCBs with different linkers in non-tumor-bearing (c) and tumor-bearing humanized mice (d). Bioavailability of active Prot-FOLR1-TCB containing different cleavage sites at day 7 after injection in non-tumor-bearing humanized NSG mice (n = 3 per group) or breast PDX tumor-bearing humanized mice (n = 6 per group). Bioavailabilities F of the active FOLR1-TCB after Prot-FOLR1-TCB administration were calculated (as described in Fig. 8) by comparing AUC 0−168 h values of FOLR1-TCB following i.v. administration of the respective pro-TCB (AUC from Prot-FOLR1-TCB) and administration of the active TCB (AUC FOLR1-TCB) according to F(%) = (AUC from Prot-TCB/AUC TCB) × 100. For tumor-bearing mice AUC were calculated from composite concentration−time data (n = 3/time point). Dose corrections were not required, as equimolar doses of Prot-FOLR1-TCB and FOLR1-TCB were used in the respective studies.