Synthesis and Biological Evaluation of Chelator-Based Small Molecule PET-Radiotracers for Imaging of PD-L1

Aim:

The programmed cell death ligand 1 (PD-L1) is overexpressed by various cancers, resulting in a downregulation of the local immune response and therefore enabling further tumor growth.[1] Immune checkpoint inhibitors (ICIs) can 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 tumor sites, there is an urgent clinical need for non-invasive diagnostic tools to support the therapeutic decision process. Small molecule-based radiotracers for noninvasive molecular PD-L1 imaging offer improved tissue penetration, fast blood clearance and low immunogenicity over radiolabeled antibodies.

Methods:

Based on a published small molecule PD-L1 inhibitor, 10 different radioligands were synthesized and radiolabeled with copper-64 (HZDR, 30 MeV TR-FLEX cyclotron). Binding affinities were determined on PC3 cells stably overexpressing human PD-L1. For in vivo evaluation, qualitative PET/CT imaging (nanoSCAN PET/CT, Mediso) was performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 xenografted tumors.

Results:

Modification of the PD-L1 binding motif with strongly water-solubilizing sulfonate and phosphonate groups, different linker units and a NODAGA-chelator in 21-25 organic synthesis steps (12-13 longest linear sequence) yielded 10 different ligands [3]. The 64Cu-labelled radiotracers exhibited logD values between -3.17 and -4.15 for six ligands of the first series, with dissociation constants (Kd) between 80.5 and 532.8 nM, as determined by saturation binding assays. Depending on the number and pattern of sulfonate and phosphonate groups, the in vivo experiments showed drastically different pharmacokinetic profiles: Compounds bearing a less hydrophilic linker showed improved tumor uptake. Three sulfonates resulted in increased blood circulation times of up to 24 h due to albumin binding increased renal clearance but also low tumor uptake (SUVmax = 1.4). Substitution of one sulfonate with a phosphonate reduced the circulation time to two hours, however, accompanied by mainly hepatobiliary clearance. To achieve predominant renal clearance, a second series of four compounds bearing two phosphonate and one sulfonate groups in the solubilizer unit and larger halogens at the central aryl core for improved tumor uptake were synthesized and are currently tested in vivo.

Conclusion:

Sulfonate groups in the PD-L1 tracers increased circulation times along with renal clearance, while tracers with one phosphonate group reduced the blood circulation time but lead to a more hepatobiliary clearance. Structural modifications to increase the binding affinity and improve tumor uptake are currently ongoing.

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] Stadlbauer S., Krutzek F., Kopka K. EP21212444.0, December 6th 2021.