PET/CT Imaging of 89Zr-N-sucDf-Pembrolizumab in Healthy Cynomolgus Monkeys

Wenping Li, Yuchuan Wang, Daniel Rubins, Idriss Bennacef, Marie Holahan, Hyking Haley, Mona Purcell, Liza Gantert, SuChun Hseih, Michael Judo, Wolfgang Seghezzi, Shuli Zhang, Elly L van der Veen, Marjolijn N Lub-de Hooge, Elisabeth G E de Vries, Jeffrey L Evelhoch, Michael Klimas, Eric D Hostetler, Wenping Li, Yuchuan Wang, Daniel Rubins, Idriss Bennacef, Marie Holahan, Hyking Haley, Mona Purcell, Liza Gantert, SuChun Hseih, Michael Judo, Wolfgang Seghezzi, Shuli Zhang, Elly L van der Veen, Marjolijn N Lub-de Hooge, Elisabeth G E de Vries, Jeffrey L Evelhoch, Michael Klimas, Eric D Hostetler

Abstract

Purpose: Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 (89Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells.

Procedures: Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis.

Results: 89Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation.

Conclusions: 89Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans.

Trial registration: ClinicalTrials.gov Identifier: NCT02760225.

Keywords: 89Zr-N-sucDf-pembrolizumab; Cynomolgus monkeys; Mesenteric lymph nodes; PD-1-positive immune cells; Positron emission tomography (PET) imaging; Spleen and tonsils; Standardized uptake values (SUVmean).

Conflict of interest statement

All authors in this manuscript were employed by either Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, or University Medical Center Groningen at the time of the study. All employees from Merck & Co., Inc. may hold/may have held stock and/or stock options.

Figures

Fig. 1.
Fig. 1.
Representative PET images of 89Zr-N-sucDf-pembrolizumab in cynomolgus monkeys. Cynomolgus monkeys were intravenously injected with tracer only or tracer co-injected with pembrolizumab at day 0. PET scans were acquired at days 0, 2, 5, and 7 following injection. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis. White arrows indicate uptake in the liver, spleen, tonsils, and mesenteric lymph node (other lymph nodes observed, but not shown in these images). (a) Animal 1 tracer-only 0.020 mg/kg. (b) Animal 2 tracer-only 0.028 mg/kg. (c) Animal 3 tracer-only 0.044 mg/kg. (d) Animal 4 co-administration of 0.210 mg/kg pembrolizumab.
Fig. 2.
Fig. 2.
PET/CT image-derived radioactivity accumulation of 89Zr-N-sucDf-pembrolizumab in cynomolgus monkeys. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis following injection of 89Zr-N-sucDf-pembrolizumab. (a) At day 0, (b) at day 2, (c) at day 5, (d) at day 7.
Fig. 3.
Fig. 3.
PET/CT image-derived radioactivity accumulation of 89Zr-N-sucDf-pembrolizumab determined as tissue to blood pool (T/B) ratio in cynomolgus monkeys. a Standardized uptake value (SUVmean) in blood pool. b SUVmean ratio of spleen to blood pool. c SUVmean ratio of lymph nodes to blood pool. d SUVmean ratio of tonsils to blood pool.
Fig. 4.
Fig. 4.
Dose-dependent decrease of 89Zr-N-sucDf-pembrolizumab tissue to blood pool (T/B) ratios in lymphoid tissues of cynomolgus monkeys at 5 days after administration of tracer and unlabeled pembrolizumab.

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