Enhancing the therapeutic range of a targeted small-molecule tubulysin conjugate for folate receptor-based cancer therapy.

Purpose: EC0305 represents a folate-tubulysin B construct capable of specifically eradicating folate receptor (FR)-positive subcutaneous tumors from mice (Leamon et al., Cancer Res 68:9839-9844, 8). Herein we report on the use of multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl-γ-glutamate) placed in-between the folate and tubulysin B moieties of EC0305 creating a new conjugate, herein referred to as EC0531, with more desirable biological properties.

Methods: The synthesis of EC0531 and its tritium-labeled counterpart are described. EC0531's affinity for FR binding and specific cytotoxic activity was assessed using standard in vitro assays. Human tumor xenografts were used to directly compare EC0305 and EC0531's antitumor activity. Finally, bile duct cannulated, female Sprague-Dawley rats were used to compare hepatobiliary clearance of these two targeted chemotherapeutic agents.

Results: EC0531 tightly binds to the FR with an affinity about half that of folic acid. It was found to specifically inhibit the growth of FR cells (IC of ~2 nM) in a dose-dependent manner. Using H-labeled compounds, more than a 12-fold higher amount of tubulysin was measured in a FR + human tumor xenograft compared to the unconjugated drug, a finding that explains, in part, why EC0531 displays curative activity, whereas the unconjugated tubulysin agent is essentially inactive. EC0531 was found to produce greater FR-specific anti-tumor activity at lower dose levels than EC0305; furthermore, EC0531's maximum tolerated dose level was significantly higher than that of EC0305, likely because EC0531's saccharopeptidic-based spacer allows for ~sixfold reduction in hepatic clearance.

Conclusions: These data provide additional evidence that the therapeutic range of targeted small-molecule drug conjugates can be favorably increased using molecular spacers constructed with 1-amino-1-deoxy-glucitolyl-γ-glutamate residues.