Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model
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It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade.
We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo.
[89Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3–5, 3.2 ± 0.4 MBq/15–16 μg/200 μl) and [64Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 μg/200 μl) were administered in mice. PET-CT scans were performed over 1–144 h ([89Zr] Keytruda) and 1–48 h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr] Keytruda), and 6.4 ± 0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr] Keytruda) and 48 h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively.
Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.
Key wordsImmunoPET Tumor-infiltrating lymphocytes PD-1 Keytruda 64-Cu 89-Zr
We would like to thank The Canary Foundation, The Ben and Catherine Ivy Foundation, and the National Cancer Institute for their support and for helping to fund this research. We acknowledge the supports of Drs. Mark Stolowitz, Timothy Doyle, Frezghi Habte, and Lingyun Xu; Sindhuja Ramakrishnan; and Michelle Tran for the experiments performed. MicroPET/CT imaging and gamma counter measurements were performed in the SCi3 Stanford Small Animal Imaging Service Center.
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Conflict of Interest
The authors declare that they have no conflict of interest.