Publications Repository - Helmholtz-Zentrum Dresden-Rossendorf

Background: Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors have the potential to reactivate the immune system, but only 30% of patients are considered responders. New diagnostic approaches re therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1 may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions. Results: Four small-molecule biphenyl-based PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. Three different chelators (NODA-GA, CB-TE2A, DiAmSar) were employed to efficiently radiolabel these compounds with copper-64, and a dimeric structure was also synthesized. All radioligands exhibited high proteolytic stability (>95%) for 48 hours post-radiolabeling. Saturation binding yielded moderate affinities ranging from 100 to 265 nM. Conversely, real-time radioligand binding revealed more promising KD values of about 20 nM for [64Cu]Cu-14 and [64Cu]Cu-15. In vivo PET imaging in mice bearing PC3 PD-L1 overexpressing and PD-L1-negative tumors was performed at 0–2, 4–5 and 24–25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator. [64Cu]Cu-14, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand [64Cu]Cu-16 demonstrated a higher liver uptake than [64Cu]Cu-15, but was still renally cleared as evidenced by the lack uptake in gall bladder and intestines. The dimeric structure [64Cu]Cu-17 showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. After 24 h post-injection, [64Cu]Cu-17 showed the highest accumulation in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs. Conclusions: This study with biphenyl-based small molecule PD-L1 radioligands clearly shows that the chelator choice significantly influences the pharmacokinetic profile. The NODA-GA-conjugated radioligand [64Cu]Cu-14 exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.