Tumor penetrating
peptides contain a cryptic (R/K)XX(R/K) CendR
element that must be C-terminally exposed to trigger
neuropilin-1 (NRP-1) binding, cellular internalization and malignant tissue penetration. The specific
proteases that are involved in processing of
tumor penetrating
peptides identified using phage display are not known. Here we design de novo a
tumor-penetrating
peptide based on consensus cleavage motif of
urokinase-type plasminogen activator (uPA). We expressed the
peptide, uCendR (RPARSGR↓SAGGSVA, ↓ shows cleavage site), on phage or coated it onto
silver nanoparticles and showed that it is cleaved by uPA, and that the cleavage triggers binding to recombinant NRP-1 and to NPR-1-expressing cells. Upon systemic administration to mice bearing uPA-overexpressing
breast tumors, FAM-labeled uCendR
peptide and uCendR-coated nanoparticles preferentially accumulated in
tumor tissue. We also show that uCendR phage internalization into cultured
cancer cells and its penetration in explants of murine
tumors and clinical
tumor explants can be potentiated by combining the uCendR
peptide with
tumor-homing module, CRGDC. Our work demonstrates the feasibility of designing
tumor-penetrating
peptides that are activated by a specific
tumor protease. As upregulation of
protease expression is one of the hallmarks of
cancer, and numerous
tumor proteases have substrate specificities compatible with proteolytic unmasking of cryptic CendR motifs, the strategy described here may provide a generic approach for designing proteolytically-actuated
peptides for
tumor-penetrative payload delivery.