Extracellularly Activatable Conjugates of RGD Peptidomimetics and Cryptophycin for α_Vβ₃-Targeted Cancer Therapy

Small molecule-drug conjugates (SMDCs) represent a promising approach in targeted cancer therapy, combining defined tumor-targeting ligands with powerful payloads. Here, we report the design, synthesis, and biological evaluation of integrin α_Vβ₃-targeting SMDCs based on a highly potent cryptophycin payload and a neutrophil elastase cleavable NPV-PABC linker. A series of stereochemically defined RGD peptidomimetics, linker epimers, and corresponding SMDCs were synthesized, providing additional negative controls for both targeting and enzymatic cleavage. The resulting conjugates remained stable in human plasma, preserved integrin-binding affinity in enzyme-linked immunosorbent assays (ELISA), and released the payload rapidly upon exposure to human neutrophil elastase depending on the stereochemistry of the linker. In vitro cytotoxicity studies in multiple cancer cell lines demonstrated that enzymatic cleavage restored cryptophycin activity to near free-drug levels, yielding picomolar potency.