Annexin A1-Targeted d-Peptide-Monomethyl Auristatin E Conjugate Enhanced Antitumor Effect of Monomethyl Auristatin E for Chemotherapy of Prostate Cancer.

Background: N-terminus 15 amino acid (MC16) of Annexin A1 (ANXA1) is an effective therapeutic target for tumors and tumor vasculature as it specifically localizes to the cell surface of tumor cells and tumor vascular endothelial cells. We identified the d-type peptide (hpnevrs, dhp7) by mirror-image phage display targeting l-type MC16 and tested the hypothesis that intravenously injected c(vcMMAE)dhp7 eradicates prostate tumor in mice with less adverse side effects than that of mc-Val-Cit-PAB-monomethyl auristatin (vcMMAE) alone.

Methods: The binding affinity of dhp7 to MC16 of ANXA1 was analyzed by biolayer interferometry and in silico conformational analysis. c(vcMMAE)dhp7 was synthesized by coupling dhp7 to vcMMAE to evaluate the antitumor effects. A subcutaneous tumor model was generated by inoculating the luciferase-expressing prostate cancer PC-3 cell line, PC-3-hLuc-PSMA, into the nude mice. 1.25 mg/kg vcMMAE or 2.14 mg/kg c(vcMMAE)dhp7, the equal dose as 1.25 mg/kg vcMMAE, was administered intravenously every 3 or 4 days and growth of tumors was assessed by photon count using an IVIS system.

Results: The dhp7 bound specifically to l-type MC16 (KD 0.48 μM) but not bound to d-type MC16 and mutant l-type MC16. Both in vitro and in silico interaction analysis indicating the α-helix of the l-type MC16 may be important for dhp7 binding. Fluorescence labeled-dhp7 accumulated in both ANXA1 positive cells and subcutaneous prostate tumor. To evaluate the antitumor effect, c(vcMMAE)dhp7 was synthesized by coupling dhp7 with mc-Val-Cit-PAB-monomethyl auristatin E (vcMMAE) as a payload. The IC50 s of vcMMAE (129.9 ng) and c(vcMMAE)dhp7 (122.7 ng) against prostate cancer cells showed almost equivalent in vitro drug sensitivity. The c(vcMMAE)dhp7-treated mice showed significant antitumor effects including complete responses at the same dose of MMAE (1.25 mg/kg) as mice treated with vcMMAE alone. Pathological analysis of the residual tumor showed that CD31 density of the c(vcMMAE)dhp7 treated group was significantly higher than that of the vcMMAE group, and the Ki-67 index was significantly lower than that of the vcMMAE alone. Clinical chemistry tests showed side effects of MMAE significantly improved in terms of hepatic/biliary toxicity in c(vcMMAE)dhp7-treated group.

Conclusions: The c(vcMMAE)dhp7 conjugate exerts a better antitumor effect than vcMMAE on ANXA1-positive prostate cancer.