Small Molecule-Based Radiotracers for PET Imaging of PD-L1 With Copper-64

Aim/Introduction
The programmed cell death ligand 1 (PD-L1) is expressed by several cancer types and leads to a downregulation of the local immune response, therefore enabling tumour cells to evade the immune response.[1] So-called immune checkpoint inhibitors (ICI) are able to reactivate the immune system, however, only 30% of the patients respond to an ICI monotherapy.[2] Since PD-L1 is heterogeneously expressed within and across tumour sites, there is an urgent clinical need for a diagnostic, non-invasive imaging probe to support therapy decision. Small molecule-based radiotracers for PD-L1 PET or SPECT imaging fulfil these requirements due to their fast clearance, low risk of side effects and highly sensitive imaging at the molecular level.[3]

Materials & Methods
Based on a published small molecule PD-L1 inhibitor, six different radioligands were synthesized and radiolabelled with copper-64 (HZDR, 30 MeV TR-FLEX cyclotron). Binding affinities were determined on PC3 cells stably overexpressing human PD-L1 which were kindly provided by the Department of Radioimmunology. For in vivo evaluation, qualitative PET/CT imaging experiments (nanoSCAN PET/CT scanner, Mediso) were performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 xenografted tumours.

Results
We designed six radioligands by modifying the PD-L1 binding motif with strongly water-solubilizing sulfonate and phosphonate groups, hydrophilic linker units and a NODAGA-chelator in 21 – 25 organic synthesis steps (12-13 longest linear sequence).[4] The copper-64 labelled radiotracers exhibited log(D) values between –3.17 and –4.15. Binding affinities (Kd) were between 80.5 and 532.8 nmol/L. Depending on the number and pattern of sulfonate and phosphonate groups, the in vivo experiments showed drastically different pharmacokinetic profiles: The radiotracer containing three sulfonates showed long circulation times of 24 h due to albumin binding, renal clearance but low tumour uptake (SUVmax = 1.4). Substitution of one sulfonate with a phosphonate improved tumour uptake (SUVmax = 3.1), reduced the circulation time to two hours but showed a more hepatobiliary clearance. The less hydrophilic radiotracer in this series with a Kd of 82.4 ± 7.42 nM, bearing one sulfonate and one phosphonate showed the most favourable pharmacokinetic profile with a short circulation time, renal clearance and an increased tumour uptake (SUVmax = 3.5).

Conclusions
The radiotracer bearing one sulfonate and one phosphonate group exhibited the best pharmacokinetic profile. This radioligand will undergo further structural modifications to increase the binding affinity and improve the tumour uptake.

References
[1] M. A. Postow, M. K. Callahan, J. D. Wolchok, J. Clin. Oncol. 2015, 33, 1974-1982.
[2] S. L. Topalian, C. G. Drake, D. M. Pardoll, Cancer Cell 2015, 27, 450-461.
[3] S. Chatterjee, W. G. Lesniak, S. Nimmagadda., Mol. Imaging 2017, 16, 1-5.
[4] S. Stadlbauer, F. Krutzek, K. Kopka, EP21212444.0, 2021.