Development of a homotrimeric PSMA radioligand based on the NOTI chelating platform

The NOTI chelating scaffold can readily be derivatized for bioconjugation without impacting its metal complexation/radiolabeling properties making it an attractive building block for the development of multimeric/-valent radiopharmaceuticals. The objective of the study was to further explore the potential of the NOTI chelating platform by preparing and characterizing homotrimeric PSMA radioconjugates in order to identify a suitable candidate for clinical translation.

Altogether, three PSMA conjugates based on the NOTI-TVA scaffold with different spacer entities between the chelating unit and the Glu-CO-Lys PSMA binding motif were readily prepared by solid phase-peptide chemistry. Cell experiments allowed the identification of the homotrimeric conjugate 9 comprising NaI-Amc spacer with high PSMA binding affinity (IC₅₀ = 5.9 nM) and high PSMA-specific internalization (17.8 ± 2.5%) compared to the clinically used radiotracer [⁶⁸Ga]Ga-PSMA-11 with a IC₅₀ of 18.5 nM and 5.2 ± 0.2% cell internalization, respectively. All ⁶⁸Ga-labeled trimeric conjugates showed high metabolic stability in vitro with [⁶⁸Ga]Ga-9 exhibiting high binding to human serum proteins (> 95%). Small-animal PET imaging revealed a specific tumor uptake of 16.0 ± 1.3% IA g^-1 and a kidney uptake of 67.8 ± 8.4% IA g^-1 for [⁶⁸Ga]Ga-9. Clinical PET imaging allowed identification of all lesions detected by [⁶⁸Ga]Ga-PSMA-11 together with a prolonged blood circulation as well as a significantly lower kidney and higher liver uptake of [⁶⁸Ga]Ga-9 compared to [⁶⁸Ga]Ga-PSMA-11.

Trimerization of the Glu-CO-Lys binding motif for conjugate 9 resulted in a ~ threefold higher binding affinity and cellular uptake as well as in an altered biodistribution profile compared to the control [⁶⁸Ga]Ga-PSMA-11 due to its intrinsic high binding to serum proteins. To fully elucidate its biodistribution, future studies in combination with long-lived radionuclides, such as ⁶⁴Cu, are warranted. Its prolonged biological half-life and favorable tumor-to-kidney ratio make this homotrimeric conjugate also a potential candidate for future radiotherapeutic applications in combination with therapeutic radionuclides such as ⁶⁷Cu.