Characterization of Site-Specifically Conjugated Monomethyl Auristatin E- and Duocarmycin-Based Anti-PSMA Antibody-Drug Conjugates for Treatment of PSMA-Expressing Tumors.

S. Lütje, D. Gerrits, J. Molkenboer-Kuenen, K. Herrmann, G. Fracasso, M. Colombatti, O. Boerman and S. Heskamp




Prostate cancer (PCa) is the most common cancer in men worldwide. In general, PCa responds poorly to chemotherapy. Therefore, antibody-drug conjugates (ADCs) have been developed to specifically deliver highly cytotoxic drugs to the tumor. Because the prostate-specific membrane antigen (PSMA) is overexpressed in PCa, it represents a promising target for ADC-based therapies. The aim of this study was to evaluate the therapeutic efficacy of site-specifically conjugated duocarmycin- and monomethyl auristatin E (MMAE)-based anti-PSMA ADCs with drug-to-antibody ratios (DARs) of 2 and 4. The glycan group of the anti-PSMA antibody D2B was chemoenzymatically conjugated with duocarmycin or MMAE. Preservation of the immunoreactivity of the antibody on site-specific conjugation was investigated in vitro. Biodistribution and small-animal SPECT/CT imaging (18.5 ± 2.6 MBq) with 25 μg of In-labeled ADCs were performed on BALB/c nude mice with subcutaneous PSMA-positive LS174T-PSMA xenografts. Finally, the therapeutic efficacy of the 4 different ADCs was assessed in mice with LS174T-PSMA tumors. The immunoreactivity of the anti-PSMA antibody was preserved on site-specific conjugation. Biodistribution revealed high tumor uptake of all agents. The highest tumor uptake was observed in mice administered with In-D2B-DAR2-MMAE, reaching 119.7 ± 37.4 percentage injected dose per gram at 3 d after injection. Tumors of mice injected with In-D2B, In-D2B-DAR2-duocarmycin, In-D2B-DAR4-duocarmycin, In-D2B-DAR2-MMAE, and In-D2B-DAR4-MMAE could clearly be visualized with small-animal SPECT/CT. In contrast to unconjugated D2B or vehicle, treatment with either of the MMAE-based ADCs, but not with a duocarmycin-based ADC, significantly impaired tumor growth and prolonged median survival from 13 d (phosphate-buffered saline) to 20 and 29 d for DAR2 and DAR4 ADC, respectively. Tumor-doubling time increased from 3.5 ± 0.5 d to 5.2 ± 1.8 and 9.2 ± 2.1 d after treatment with D2B-DAR2-MMAE and D2B-DAR4-MMAE, respectively. The site-specifically conjugated anti-PSMA ADCs D2B-DAR2-MMAE and D2B-DAR4-MMAE efficiently targeted PSMA-expressing xenografts, effectively inhibited tumor growth of PSMA-expressing tumors, and significantly prolonged survival of mice.