Comparative evaluation of 225 Ac-PSMA-Trillium with other 225 Ac-PSMA-SMOL assets in a preclinical prostate cancer xenograft model

Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein highly expressed on prostate cancer cells, making it an ideal target for novel prostate cancer therapies. Currently, there are several actinium-225 ( 225 Ac)-PSMA-targeting small molecules (SMOL) in clinical development for treating patients with metastatic castration-resistant prostate cancer. 225 Ac-PSMA-Trillium (BAY 3563254), is a novel PSMA-targeting molecule which comprises a highly specific PSMA-binding motif, an albumin-binding domain to optimize tumor uptake and retention, and a Macropa™ chelator complexed with the alpha-emitter 225 Ac. Here, we compared 225 Ac-PSMA-Trillium with three other 225 Ac-PSMA-SMOLs, i.e., 225 Ac-PSMA-617, 225 Ac-PSMA-R2 and 225 Ac-PSMA-I&T, which all utilize a DOTA chelator for complexing 225 Ac. All assets were synthesized and radiolabeled at Bayer AG. Binding to PSMA was confirmed using a surface plasmon resonance (SPR) assay . In vitro cytotoxicity in LNCaP cells was evaluated using CellTiterGlo®. The antitumor effects of 225 Ac-PSMA-SMOLs were investigated in male SCID mice with subcutaneous LNCaP tumors. Mice were randomized and treated with a single i.v injection of 225 Ac-PSMA-Trillium or the other 225 Ac-PSMA-SMOLs at different dose levels (100-400 kBq/kg, 375 kBq/nmol). Tumor growth and body weights were monitored for 4 weeks post injection. 225 Ac activity in various organs and tumors was measured using a high purity germanium detector or autoradiography. 177 Lu-PSMA-617 SPECT imaging was performed to evaluate tumor uptake. All PSMA-SMOLs exhibited strong binding affinity to recombinant PSMA as demonstrated by SPR. When radiolabeled with non-radioactive lanthanum as surrogate for 225 Ac, the target residence time of PSMA-SMOLs was weakened, except for PSMA-Trillium. All 225 Ac-labeled assets induced potent cytotoxicity in LNCaP cells in vitro. Radio-HPLC and iTLC confirmed the integrity and high radiochemical purity, respectively. 225 Ac-PSMA-Trillium at 400 kBq/kg showed higher tumor uptake compared to other 225 Ac-PSMA-SMOLs, homogeneous tumor accumulation 60 hours after injection, and fast clearance from the kidneys based on autoradiography. Furthermore, 225 Ac-PSMA-Trillium at all doses inhibited LNCaP tumor growth and decreased tumor weights in a dose-dependent manner, whereas the antitumor response was restricted to the higher doses for all other compounds. In summary, 225 Ac-PSMA-Trillium showed the highest tumor uptake and the strongest tumor growth inhibition with a clear dose response compared with the other 225 Ac-PSMA-SMOLs. These data support the clinical development of 225 Ac-PSMA-Trillium (NCT06217822).