Gene targeting strategy for B-NDG Ces1c KO mice. The exons 2-13 of mouse Ces1c were knocked out in B-NDG Ces1c KO mice.

Strain specific Ces1c expression analysis in homozygous B-NDG Ces1c KO mice by Western blot. Liver and serum were collected from B-NDG mice (+/+) and homozygous B-NDG Ces1c KO mice (-/-), and then analyzed by western blot with species-specific anti-Ces1c antibody. 40 μg total proteins were loaded for western blotting analysis. Ces1c was not detected in liver and serum of homozygous B-NDG Ces1c KO mice (-/-).

In vitro analysis of the kinetics of Ab1-vcMMAE (in-house) in B-NDG Ces1c KO mice. Ab1-vcMMAE (in-house) was added to B-NDG mice, B-NDG Ces1c KO mice, human plasma, and 0.5% BSA at a concentration of 100 µg/mL and incubated at 37°C for 0h, 1d, 3d, 8d, and 14d. The free MMAE content was assessed at each time point. At 14 days, the release rate of free MMAE in B-NDG mice was approximately 30%, while the release rates in B-NDG Ces1c KO mice were similar to those in human plasma and 0.5% BSA. This indicates that B-NDG Ces1c KO mice provide a good pharmacokinetic/pharmacodynamic model for ADC drugs that are susceptible to cleavage by Ces1c.