A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins.

Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called "G4") that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.