Abstract View

Objectives

PSMA-targeted radioligand therapy is an established strategy for metastatic prostate cancer; however, limited treatment response and therapy resistance remain clinical challenges¹. Increasing the achievable molar activity may allow higher activity to be delivered with lower ligand mass, while preserving tumor specificity. Therefore, this study investigated whether introducing multiple chelators into clinically validated PSMA ligand scaffolds could improve ¹⁷⁷Lu radiolabeling efficiency, increase achievable molar activity, and enhance biological performance.

Methods

Novel PSMA ligands, including PSMA-617-2D, PSMA-617-3D and PSMA I&T-2D, were synthesized by solid-phase peptide synthesis and characterized by RP-HPLC, ESI-MS and MALDI-MS. Radiolabeling with ¹⁷⁷Lu was evaluated at 40–95 °C over time, and radiochemical purity was assessed by radio-HPLC. Molar activity capacity was investigated by increasing activity-to-ligand ratios. Lipophilicity was determined using an octanol/PBS system. Stability was evaluated in PBS and human serum. PSMA affinity, binding and internalization were studied in PSMA-positive PC3-PIP and LNCaP, and PSMA-negative PC3-Flu cells. Preliminary in vivo performance was assessed by SPECT/CT imaging in LNCaP tumor-bearing SCID mice.

Results

The multi-chelator strategy improved ¹⁷⁷Lu radiolabeling performance. At 60 °C, [¹⁷⁷Lu]Lu-PSMA-617-2D reached >90% radiochemical purity within 30 min, whereas the parent [¹⁷⁷Lu]Lu-PSMA-617 required up to 60 min under comparable conditions. At 40 °C, PSMA-617-2D showed faster labeling than PSMA-617 and behaved similarly to double-concentrated PSMA-617, supporting the hypothesis that increased chelator availability accelerates complexation. PSMA-617-3D was also successfully radiolabeled, but the third chelator did not provide further improvement over PSMA-617-2D, suggesting a plateau beyond two chelating units.

Importantly, both 2D and 3D derivatives showed increased molar activity capacity compared with PSMA-617. At high activity-to-ligand ratios, the parent ligand rapidly lost labeling efficiency, whereas the multi-chelator derivatives maintained substantially higher radiolabeling performance. This indicates that additional chelators can increase the amount of ¹⁷⁷Lu incorporated per targeting vector, which could potentially reduce the number of required patient doses.

All compounds remained strongly hydrophilic, with LogD values of approximately −3, while PSMA affinity was preserved in the low nanomolar range, with IC₅₀ values of 8–29 nM. Stability studies showed good overall integrity, with PSMA-617 and PSMA-617-2D displaying the highest radiochemical stability over time. The additional chelator did not compromise PSMA-specific uptake; instead, [¹⁷⁷Lu]Lu-PSMA-617-2D showed enhanced internalization in PC3-PIP cells compared with the parent compound, corresponding to 72% versus 58% of total uptake. In vivo SPECT/CT imaging confirmed clear tumor visualization. At 24 h p.i., [¹⁷⁷Lu]Lu-PSMA-617-2D showed the highest tumor SUV_max value of 15.1, compared with 11.1 for [¹⁷⁷Lu]Lu-PSMA-617 and 11.5 for [¹⁷⁷Lu]Lu-PSMA I&T.

Conclusions

Introducing two chelators into PSMA-617 improved radiolabeling kinetics, molar activity capacity and biological performance while preserving hydrophilicity, stability and PSMA affinity. PSMA-617-2D emerged as the lead candidate, showing faster ¹⁷⁷Lu complexation, enhanced internalization and increased tumor SUV_max. These findings support two-site chelation as a practical strategy for developing next-generation PSMA-targeting radiopharmaceuticals with higher radioactive payload and improved therapeutic potential.

References

Kostos, L. et al. (2023). Determinants of outcome following PSMA-based radioligand therapy and mechanisms of resistance in patients with metastatic castration-resistant prostate cancer. Therapeutic Advances in Medical Oncology. 15.