Targeting therapy is a concept that has gained significant importance in recent years, especially in oncology. The severe dose-limiting side effects of chemotherapy necessitate the development of novel, efficient and tolerable therapy approaches. In this regard, the prostate specific membrane antigene (PSMA) has been well established as a molecular target for diagnosis of, as well as therapy for, prostate cancer. Although most PSMA-targeting ligands are radiopharmaceuticals used in imaging or radioligand therapy, this article evaluates a PSMA-targeting small molecule–drug conjugate, and, thus, addresses a hitherto little-explored field. PSMA binding affinity and cytotoxicity were determined in vitro using cell-based assays. Enzyme-specific cleavage of the active drug was quantified via an enzyme-based assay. Efficacy and tolerability in vivo were assessed using an LNCaP xenograft model. Histopathological characterization of the tumor in terms of apoptotic status and proliferation rate was carried out using caspase-3 and Ki67 staining. The binding affinity of the Monomethyl auristatin E (MMAE) conjugate was moderate, compared to the drug-free PSMA ligand. Cytotoxicity in vitro was in the nanomolar range. Both binding and cytotoxicity were found to be PSMA-specific. Additionally, complete MMAE release could be reached after incubation with cathepsin B. In vivo, the MMAE conjugate displayed good tolerability and dose-dependent inhibition of tumor growth. Immunohistochemical and histological studies revealed the antitumor effect of MMAE.VC.SA.617, resulting in the inhibition of proliferation and the enhancement of apoptosis. The developed MMAE conjugate showed good properties in vitro, as well as in vivo, and should, therefore, be considered a promising candidate for a translational approach.

(1) Background: Prostate-specific membrane antigen (PSMA) has been extensively studied in the last decade. It became a promising biological target in the diagnosis and therapy of PSMA-expressing cancer diseases. Although there are several radiolabeled PSMA inhibitors available, the search for new compounds with improved pharmacokinetic properties and simplified synthesis is still ongoing. In this study, we developed PSMA ligands with two different hybrid chelators and a modified linker. Both compounds have displayed a promising pharmacokinetic profile. (2) Methods: DATA^5m.SA.KuE and AAZTA⁵.SA.KuE were synthesized. DATA^5m.SA.KuE was labeled with gallium-68 and radiochemical yields of various amounts of precursor at different temperatures were determined. Complex stability in phosphate-buffered saline (PBS) and human serum (HS) was examined at 37 °C. Binding affinity and internalization ratio were determined in in vitro assays using PSMA-positive LNCaP cells. Tumor accumulation and biodistribution were evaluated in vivo and ex vivo using an LNCaP Balb/c nude mouse model. All experiments were conducted with PSMA-11 as reference. (3) Results: DATA^5m.SA.KuE was synthesized successfully. AAZTA⁵.SA.KuE was synthesized and labeled according to the literature. Radiolabeling of DATA^5m.SA.KuE with gallium-68 was performed in ammonium acetate buffer (1 M, pH 5.5). High radiochemical yields (>98%) were obtained with 5 nmol at 70 °C, 15 nmol at 50 °C, and 60 nmol (50 µg) at room temperature. [⁶⁸Ga]Ga-DATA^5m.SA.KuE was stable in human serum as well as in PBS after 120 min. PSMA binding affinities of AAZTA⁵.SA.KuE and DATA^5m.SA.KuE were in the nanomolar range. PSMA-specific internalization ratio was comparable to PSMA-11. In vivo and ex vivo studies of [¹⁷⁷Lu]Lu-AAZTA⁵.SA.KuE, [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE and [⁶⁸Ga]Ga-DATA^5m.SA.KuE displayed specific accumulation in the tumor along with fast clearance and reduced off-target uptake. (4) Conclusions: Both KuE-conjugates showed promising properties especially in vivo allowing for translational theranostic use.