In Vivo Evaluation and Dosimetry Estimate for a High Affinity Affibody PET Tracer Targeting PD-L1

In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [¹⁸F]AlF-NOTA-Z_{PD-L1_1} as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule Z_{PD-L1_4}, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1.

Z_{PD-L1_4} was conjugated with NOTA and radiolabeled with either [¹⁸F]AlF or ⁶⁸Ga. [¹⁸F]AlF-NOTA-Z_{PD-L1_4} and [⁶⁸Ga]NOTA-Z_{PD-L1_4} were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses.

Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([¹⁸F]AlF-NOTA-Z_{PD-L1_4}: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [⁶⁸Ga]NOTA-Z_{PD-L1_4}: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than Z_{PD-L1_1}. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([¹⁸F]AlF-NOTA-Z_{PD-L1_4}: 1.26 ± 0.13 SUV, [⁶⁸Ga]NOTA-Z_{PD-L1_4}: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses.

[¹⁸F]AlF-NOTA-Z_{PD-L1_4} and [⁶⁸Ga]NOTA-Z_{PD-L1_4} are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [¹⁸F] or [⁶⁸Ga] may expand access to clinical sites.