In Vivo Imaging of the Programmed Death Ligand 1 by ¹⁸F PET

Abstract

Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-Z with F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. The affinity of the PD-L1-binding Affibody ligand Z was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of F-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by γ-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. NOTA-Z was labeled, with a radiochemical yield of 15.1% ± 5.6%, radiochemical purity of 96.7% ± 2.0%, and specific activity of 14.6 ± 6.5 GBq/μmol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 ± 0.33) and -negative (%ID/g = 0.32 ± 0.05) tumors ( = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.

Noninvasive imaging of the PD-1:PD-L1 immune checkpoint: Embracing nuclear medicine for the benefit of personalized immunotherapy

“…Uptake in non-tumor tissues was low, with the highest signals in kidney and bladder, reflecting excretion, but also in liver, which showed non-negligible uptake of the radiotracer. Ex vivo autoradiography of mixed PD-L1 pos and PD-L1 neg tumors showed heterogeneous uptake, with areas of highest radiotracer uptake corresponding to PD-L1 positivity on IHC 70 .…”

Affibody Molecules as Targeting Vectors for PET Imaging

“…Affinity maturation permits a further increase of the affinity from a few nM to a few pM. High-affinity affibody binders have been selected for potential cancer-associated molecular targets such as human epidermal growth factor receptor type 2 (HER2) [22], epidermal growth factor receptor (EGFR or HER1) [23], human epidermal growth factor receptor type 3 (HER3) [24], insulinlike growth factor-1 receptor (IGF-1R) [25], platelet-derived growth factor receptor β (PDGFRβ) [26], vascular endothelial growth factor receptor 2 (VEGFR2) [27], programmed death-ligand 1 (PD-L1) [28], and carbonic anhydrase IX (CAIX) [29]. Several features of affibody molecules make them potentially attractive as targeting vectors for imaging probes:…”

“…[ 18 F]-AlF-NOTA-ZHER2:2395 demonstrated excellent tumor targeting 4 h pi (tumor-to-blood ratio of 145 ± 24), but the bone uptake was twice as high as that of its 111 In-labeled counterpart, which indicates a somewhat lower chelate stability. This approach has been applied to label the HER2-binding affibody molecules NOTA-CGGGRDN-ZHER2:234 [86], and NOTA-ZHER2:2891-C [58], PD-L1-binding NOTA-ZPD-L1_1 [28], and EGFR-binding NOTA-ZEGFR:1907 [87] and NOTA-ZEGFR:03115 [88], providing radiochemical yields in the range of 10-15% after a 40-min procedure. Another efficient approach for improving the radiofluorination yield is based on the formation of [ 18 F]aluminium monofluoride ([ 18 F]-AlF), with subsequent complexation by derivatives of the NOTA chelator [85].…”

Role of medical imaging for immune checkpoint blockade therapy: From response assessment to prognosis prediction

“…Despite the tremendous potential of antibody‐based tracers, they have slow blood clearance that leads to long waiting time (eg, several days) between imaging scan and tracer injection, which prevents the clinical transformation of the molecular imaging. In this setting, a number of protein‐ or peptide‐based radiotracers have been fabricated to compensate the pitfalls of the antibody‐based probes . For example, Trotte et al developed a 18 F‐labeled affibody ligand that can effectively detect PD‐L1 expression in xenograft tumors by PET imaging, with preferable specificity, fast blood clearance, and low normal tissue uptake except nephridia .…”

“…In this setting, a number of protein‐ or peptide‐based radiotracers have been fabricated to compensate the pitfalls of the antibody‐based probes . For example, Trotte et al developed a 18 F‐labeled affibody ligand that can effectively detect PD‐L1 expression in xenograft tumors by PET imaging, with preferable specificity, fast blood clearance, and low normal tissue uptake except nephridia . Larimer et al demonstrated that granzyme B PET imaging might serve as an effective biomarker for predicting responses to immunotherapy in human tumor xenograft models …”