Preclinical evaluation of new PET tracers for in vivo imaging of PD-L1 expression

Introduction: PD-L1 expression in some tumor types correlates with therapeutic efficacy of PD-1 inhibitors. In vivo imaging of PD-L1 could potentially monitor changing PD-L1 expression and heterogeneity of PD-L1 expression within and across tumors in a subject. Previously, we evaluated the PD-L1-targeting Affibody molecule ZPD-L1_1 as a potential PET tracer in a mouse tumor model of human PD-L1 expression (González Trotter, et al., 2017). In this study, we performed a similar evaluation with the affinitymatured Affibody molecule ZPD-L1_4, to determine if the greater affinity for PD-L1 of ZPD-L1_4 would result in improved in vivo targeting of PD-L1. Materials and methods: The binding affinity of ZPD-L1_4 for human PD-L1 was measured using Surface Plasmon Resonance (SPR). For imaging, ZPD-L1_4 was conjugated with NOTA, and radiolabeled with either [18F]AlF or 68Ga. Both PET tracers were evaluated in SCID Beige mice with LOX (hPD-L1+) and SUDHL6 (hPD-L1-) tumors. PET data were acquired for 90 minutes following I.V. administration of the tracer. Immediately after imaging, mice were euthanized for ex vivo biodistribution measurements. Results: ZPD-L1_4 demonstrated very high affinity for human PD-L1 (apparent KD = 0.07 nM). [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTAZPD- L1_4 were both successfully synthesized: [18F]AlF-NOTA-ZPD-L1_4: SA 330-460 Ci/mmol @EOS; 100% RCP and [68Ga]NOTA-ZPD-L1_4: SA 240- 250 Ci/mmol @EOS; 100% RCP. PET imaging showed similar pharmacokinetics and clearance for both tracers. LOX tumors were clearly visible in PET images. Renal clearance was the primary route of elimination for both tracers. Ex vivo biodistribution measurements showed that both tracers had >25 fold higher accumulation in LOX tumors than SUDHL6: [18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6) and [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6). For comparison, for [18F]AlF-NOTAZPD- L1_1, LOX tumor uptake was < 3 %ID/g. Blood and plasma accumulation measurements were > 3 fold higher for [68Ga]NOTA-ZPD-L1_4 than [18F] AlF-NOTA-ZPD-L1_4, and very high kidney accumulation was measured for both tracers. Conclusions: ZPD-L1_4 had markedly higher apparent affinity for human PD-L1 than the previously evaluated ZPD-L1_1, and demonstrated improved targeting of PD-L1 in a mouse model. The development of both 18F- and 68Ga-labeled PET tracers may expand access to clinical sites, as many sites can only produce one o