PROJECT SUMMARY The goal of this proposal is to develop a targeted therapy for treatment of metastatic castration-resistant prostate cancer (mCRPC). There is currently no cure for this disease and available therapies leave significant room for improvement based on dismal survival rates. Prostate-specific membrane antigen (PSMA) is a clinically validated molecular target for diagnostic and therapeutic purposes based on its significant overexpression in PCa tissue compared to normal cells. Glutamate-ureido-based small molecules have exceptional affinity for PSMA and have been labeled with radionuclides to enable targeted tumor detection and therapy. The small molecule 177Lu- PSMA-617 (cytotoxic beta radiation emitter) recently gained FDA-approval for PSMA-targeted radioligand therapy (RLT) of mCRPC. However, despite its initial antitumor activity, disease progression following 177Lu- PSMA-617 RLT occurs in virtually all patients and led to the development of the more potent RLT agent 225Ac- PSMA-617, where the alpha-emitting radionuclide 225Ac provides stronger DNA damaging capabilities. Although recent clinical data with 225Ac-PSMA-617 showed favorable responses, disease progression was still observed. Furthermore, non-tumor binding of 225Ac-PSMA-617 causes considerable damage to the salivary glands and leads to life-long xerostomia in a large percentage of patients. Since PSMA expression often remains actionable as a tumor target following RLT, the development of a non-radioactive targeted therapy could extend the therapeutic benefits of PSMA targeting without the associated risk of organ irradiation. Given the absence of a validated PSMA-targeted drug conjugate, we used PSMA-617 as the foundation for a drug conjugate containing the microtubule inhibitor monomethyl auristatin E (MMAE) and a custom macrocyclic linker known as MMC (multimodality chelator) to enable direct radiolabeling for imaging and quantitative analyses. We provide strong preliminary evidence of PS