Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model

Abstract

Purpose: 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.

Procedures: 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-2Rγ^null (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.

Results: [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.

Conclusions: 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.

Keywords: 64-Cu; 89-Zr; ImmunoPET; Keytruda; PD-1; Tumor-infiltrating lymphocytes.