In vivo PK Study

All animal procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Buffalo. Ocular samples were collected alongside the studies carried out in Li et al. (2019) in MDA-MB-468 tumor bearing nude mice (33). Trastuzumab, F(ab)₂, Fab, and scFv were each administered by penile vein injection at a dose of 10 mg/kg. Four terminal sampling time points of three mice each were selected for each protein. For trastuzumab, samples were collected at 6, 24, 72, and 168 h. For F(ab)₂, samples were collected at 1, 6, 24, and 48 h. For Fab, samples were collected at 10 min and 1, 4, and 24 h. For scFv, samples were collected at 5 min and 1, 4, and 24 h. The T-vc-MMAE PK study was carried out separately in N87 tumor bearing nude mice that received a 10 mg/kg dose of the ADC by penile vein injection. Terminal samples were collected at 10 min and 6, 24, 72, and 168 h. At each time point, blood was collected by cardiac puncture, and plasma separated by centrifugation (2000 g for 20 min) and stored at − 80 °C. Both eyes were enucleated as described above and stored in individual microcentrifuge tubes at − 80 °C until dissection.