INTRODUCTION:Tau-positron emission tomography (PET) outcome data of patients with Alzheimer's disease (AD) cannot currently be meaningfully compared or combined when different tracers are used due to differences in tracer properties, instrumentation, and methods of analysis. METHODS:Using head-to-head data from five cohorts with tau PET radiotracers designed to target tau deposition in AD, we tested a joint propagation model (JPM) to harmonize quantification (units termed "CenTauR" [CTR]). JPM is a statistical model that simultaneously models the relationships between head-to-head and anchor point data. JPM was compared to a linear regression approach analogous to the one used in the amyloid PET Centiloid scale. RESULTS:A strong linear relationship was observed between CTR values across brain regions. Using the JPM approach, CTR estimates were similar to, but more accurate than, those derived using the linear regression approach. DISCUSSION:Preliminary findings using the JPM support the development and adoption of a universal scale for tau-PET quantification. HIGHLIGHTS:Tested a novel joint propagation model (JPM) to harmonize quantification of tau PET. Units of common scale are termed "CenTauRs". Tested a Centiloid-like linear regression approach. Using five cohorts with head-to-head tau PET, JPM outperformed linearregressionbased approach. Strong linear relationship was observed between CenTauRs values across brain regions.

MK-8189 is a novel phosphodiesterase 10A (PDE10A) inhibitor being evaluated in clinical studies for the treatment of schizophrenia. PDE10A is a cyclic nucleotide phosphodiesterase enzyme highly expressed in medium spiny neurons of the striatum. MK-8189 exhibits subnanomolar potency on the PDE10A enzyme and has excellent pharmaceutical properties. Oral administration of MK-8189 significantly increased cyclic guanosine monophosphate and phospho glutamate receptor 1 in rat striatal tissues. Activation of the dopamine D1 direct and D2 indirect pathways was demonstrated by detecting significant elevation of mRNA encoding substance P and enkephalin after MK-8189 administration. The PDE10A tracer [3H]MK-8193 was used to determine the PDE10A enzyme occupancy (EO) required for efficacy in behavioral models. In the rat-conditioned avoidance responding assay, MK-8189 significantly decreased avoidance behavior at PDE10A EO greater than ∼48%. MK-8189 significantly reversed an MK-801-induced deficit in prepulse inhibition at PDE10A EO of ∼47% and higher. Target engagement of MK-8189 in rhesus monkeys was examined with [11C]MK-8193 in positron emission tomography studies, and plasma concentrations of 127 nM MK-8189 yielded ∼50% EO in the striatum. The impact of MK-8189 on cognitive symptoms was evaluated using the objective retrieval task in rhesus monkeys. MK-8189 significantly attenuated a ketamine-induced deficit in object retrieval performance at exposure that yielded ∼29% PDE10A EO. These findings demonstrate the robust impact of MK-8189 on striatal signaling and efficacy in preclinical models of symptoms associated with schizophrenia. Data from these studies were used to establish the relationship between preclinical efficacy, plasma exposures, and PDE10A EO to guide dose selection of MK-8189 in clinical studies. SIGNIFICANCE STATEMENT: We describe the primary pharmacology of MK-8189, a phosphodiesterase 10A (PDE10A) inhibitor under evaluation for the treatment of schizophrenia. We report efficacy in preclinical models that have been used to characterize other PDE10A inhibitors and atypical antipsychotics. The PDE10A occupancy achieved by MK-8189 in behavioral studies was used to support dose selection in clinical trials. This work provides evidence to support exploration of higher levels of PDE10A occupancy in clinical trials to determine if this translates to improved efficacy in patients.

Tau positron emission tomography (PET) is increasingly used in the clinical evaluation of patients and as an outcome measure in Alzheimer’s disease (AD) clinical trials. Due to differences in tracer properties, instrumentation, and methods of analysis, however, tau-PET outcome data cannot currently be meaningfully compared or combined. Here, we tested i) the feasibility of adapting the Centiloid method—an approach originally developed to standardize amyloid PET—to harmonize tau-PET quantification (CenTauRs); ii) the performance of a non-linear mixed model-based approach (Joint Propagation Model) that does not require the use of a reference tracer. Head-to-head tau-PET data (Table 1) was included from two cohorts ([ 18 F]RO948 vs [ 18 F]flortaucipir, n = 37 [BioFINDER-2]; [ 18 F]flortaucipir [Avid A05] vs [ 18 F]MK-6240, n = 15, University of Pittsburgh) in which each participant was scanned with two tau-PET tracers. Standardized uptake value ratio (SUVR) values were calculated using the inferior cerebellar cortex as the reference region. Anchor point data (Table 1) was derived for each tracer using the following criteria: CenTauR-0, cognitively unimpaired (CU), amyloid PET negative (<10 Centiloids); CenTauR-100, amyloid PET positive (>50 Centiloids), typical (temporoparietal) AD pattern on tau-PET visual read, age<65 andMMSE>20. Regions-of-interest (ROIs) included a universal tau-PET ROI—based on the intersection of tracer specific ([ 18 F]flortaucipir, [ 18 F]MK-6240, [ 18 F]PI-2620, [ 18 F]PM-PBB3, [ 18 F]GTP1 and [ 18 F]RO948) masks that had been derived by subtracting average of amyloid-negative CU images from the average AD image—as well as four subregions delineated within this ROI (medial temporal, meta-temporal, temporoparietal and frontal) (Figure 1A). An overview of the adapted Centiloid-like approach and the joint propagation model is shown in Figure 1B. High R 2 values were observed between tracers across all ROIs: [ 18 F]RO948 vs [ 18 F]flortaucipir, average 0.965 [range 0.923 (frontal) to 0.986 (universal)]; [ 18 F]MK-6240 vs [ 18 F]flortaucipir, average 0.985 [range, 0.923 (medial temporal) to 0.991 (frontal)]. The Centiloid-like and joint propagation model approaches provided near identical CenTauR values (Figure 2). Preliminary findings support the development of standardized scale for tau-PET using both a Centiloid-like or joint propagation model approach. Additional data and consideration of the advantages and disadvantages of each will be needed to recommend one approach over the other.

[ 18 F]MK6240 is a second-generation radiotracer for imaging tau pathology in vivo. Regional thresholds that allow for an assessment of Braak stage have yet to be established and importantly may vary between regions. In this work we use a Z-score approach to define regional thresholds and estimate Braak stage in Alzheimer’s Disease. All static MK6240 images used were downloaded from the Lantheus/Cerveau database, spatially normalised into MNI space using DARTEL and SUVR images were computed using an inferior cerebellum reference region. Three Braak meta-ROIS, Braak I/II, Braak III/IV and Braak V/VI (Figure 1), were applied to 275 amyloid negative healthy control images to define regional thresholds for positivity in each ROI at the level of mean SUVR + 2 SD. These thresholds were then applied to 232 scans from subjects across the AD spectrum with associated MMSE data (88 healthy control, 68 MCI and 76 AD: 131 Aβ+ / 101 Aβ-). [ 18 F]MK6240 showed similar mean values and low variability across all ROIs in the amyloid negative healthy controls (Figure 1). The regional SUVR cut-off values were estimated as: Braak I/II = 1.29, Braak III/IV = 1.15 and Braak V/VI = 1.15. When these cut-offs were applied to subjects across the AD spectrum with MMSE data (Figure 2), 118 out of 232 were negative for Braak pathology. These subjects were predominantly healthy controls and their mean MMSE was 27.3. For subjects that demonstrated Braak positivity in at least one region, 104 subjects demonstrated an ordering consistent with Braak staging whilst 10 subjects had an atypical ordering consistent with hippocampal sparing. 64 subjects were positive in all Braak regions with a mean MMSE of 23.4 and all but one of these subjects was either MCI or AD. Analysis of [ 18 F]MK6240 images with thresholds that allow for the determination of Braak stage status provides data that are consistent with clinical status and MMSE score. This approach may be useful in natural history studies and clinical trials.

Deposition of misfolded α-synuclein (α-Syn) aggregates in the human brain is one of the major hallmarks of synucleinopathies. Positron Emission Tomography imaging (PET) of α-synuclein in Parkinson’s disease (PD) patients is highly desirable but remains elusive. An extensive PET radioligand discovery campaign was undertaken. Here, we present the in-vitro and preclinical in-vivo characterization of [ 3 H]- and [ 11 C]Compound S, representing an example of a promising PET radioligand for α-Syn. Competition binding studies and autoradiographic studies were performed in cortexes of PD patients, healthy control, and Alzheimer’s disease (AD) patients brain tissue, and mid-brain region of the aged A30P mouse using [ 3 H]S. Brain PET experiments using [ 11 C]S were carried out in a healthy non-human primate (NHP) and the aged homozygote A30P mice. Compound S is a sub-nanomolar affinity ligand to human and A30P mouse α-Syn (Kd = 0.15 nM and 0.5 nM resp.). Lower affinity was observed in AD and healthy control brain tissues. [ 3 H]S showed displaceable binding in PD brain cortex and A30P mid-brain and brainstem, and little binding in AD and healthy control. In the NHP, [ 11 C]S crosses the brain-blood barrier and the uptake phase of [ 11 C]S is followed by a washout. [ 11 C]S PET studies in A30P mice showed higher uptake in the mid brain and brainstem regions, with standard uptake value ratios in those regions higher than 2.0. Compound S is potent for α-Syn in vitro, and PET imaging in the A30P mice showed an elevated signal in regions known to accumulate α-Syn. This dataset suggests that the Compound S has promise towards imaging α-Syn in PD patients .

Modification of potent, selective metabotropic glutamate receptor 2 negative allosteric modulator (mGluR2 NAM) led to a series of analogues with excellent binding affinity, lipophilicity, and suitable physicochemical properties for a PET tracer with convenient chemical handles for incorporation of a 11C or 18F radiolabel. [11C]MK-8056 was synthesized and evaluated in vivo and demonstrated appropriate affinity, selectivity, and physicochemical properties to be used as a positron emission tomography tracer for mGluR2.

Immunotherapy has revolutionized the way cancer is treated; however, despite tremendous success, not all patients benefit from immunotherapy. Identifying biomarkers predictive of effective treatment response to current and novel immunotherapies is of utmost importance. Multiple studies have indicated neutrophil-lymphocyte ratio (NLR) may be a predictive biomarker of immunotherapy response. We analyzed hematological parameters across multiple preclinical murine tumors to determine if NLR predicts tumor growth and response to treatment. The hematological survey identified NLR to be strongly correlated to tumor weights, with larger tumors exhibiting higher NLRs. In addition, mouse tumors that have been characterized as resistant or partially responsive to immunotherapy had a higher NLR than tumors responsive to immunotherapy. To track neutrophils in vivo, we generated a BAC transgenic mouse wherein firefly luciferase (Luc2), diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) genes were expressed under the endogenous Ly6G promoter. The Ly6G promoter enables tracking of Ly6G+ neutrophils using luciferase-based bioluminescence imaging (BLI), depletion of Ly6G+ neutrophils by diphtheria toxin treatment, and the ability to perform flow analyses using the eGFP marker. In vivo and ex vivo BLI demonstrated that the Ly6G+ cells were primarily expressed in bone marrow, blood, spleen, and the digestive tract. To ensure the specificity of the bioluminescence signal, we isolated Ly6G+ and Ly6G- immune cells and performed in vitro BLI. In vitro BLI confirmed that bioluminescence was exclusive to Ly6G+ cells. To confirm that the Luc2-DTR-eGFP (LDG) reporter cassette expression did not affect neutrophil activity and function, we quantified the level of myeloperoxidase (MPO), a marker of neutrophil activation, in the phorbol myristate acetate (PMA)-induced ear edema mouse model. Neutrophil activity was measured using luminol enabled BLI of MPO. Luminol-BLI following PMA-induced ear edema demonstrated similar MPO activity between wildtype and transgenic mice. Finally, we investigated if the LDG reporter cassette impacted tumor growth. Measures of tumor burden in this transgenic line recapitulated our hematological findings. Tumors with higher NLR showed higher tumor BLI signal when compared to tumors with lower NLR ratio, demonstrating that the LDG reporter cassette did not impact tumor growth in the transgenic mice and importantly, providing a tool to track Ly6G+ cells in tumors. Our results demonstrate that NLR in tumor correlates with response to immunotherapy across multiple tumor types. Here we describe the development of a transgenic mouse to track intratumoral Ly6G+ cells. These transgenic mice provide a valuable tool for profiling oncology compounds, including those targeting immune cells, and for understanding the mechanism of action of such agents. Citation Format: Milind D. Chalishazar, Nicolas Solban, Johnny Kopinja, Doug Linn, Razvan Cristescu, Heather Zhou, Thomas Rosahl, Brian Long, Weisheng Zhang, Eric Hostetler. Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2455.

MK-8189 is an investigational selective PDE10A inhibitor for treating schizophrenia that reduces striatal D2 and increases D1 and NMDA signaling in preclinical models.

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer’s disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

The muscarinic acetylcholine receptor 4 (M4) has attracted attention as a target for treating neuropsychiatric symptoms, such as agitation and aggression, associated with Alzheimer’s disease. We have developed novel selective, potent, and in vivo suitable M4 positive allosteric modulators (PAMs) and demonstrated robust pharmacology in preclinical models predictive of antipsychotic and pro-cognitive effects. Importantly, we have also shown that these selective M4 PAMs produce greater therapeutic margins for cholinergic adverse effects compared to less selective muscarinic receptor agonists. To enable translation in the clinic, we set out to discover an M4 PAM PET tracer suitable for measuring central receptor occupancy (RO). [11C]MK-6884 binds with high affinity and selectivity to the allosteric site on the M4 receptor that our other M4 PAMs bind. The affinity of [11C]MK-6884 was found to be influenced by the concentration of orthosteric agonist in vitro and in vivo, consistent with the high cooperativity of the molecule in vitro. PET studies successfully determined the target engagement of PAMs to enable modeling the relationship between drug plasma exposure, RO, and pharmacodynamic activity. Donepezil increased the tracer binding potential of [11C]MK-6884 in rhesus and healthy human subjects, confirming in vivo the influence of cholinergic tone on [11C]MK-6884 binding that was observed in vitro. Consistent with a presumed cortical deficit in synaptic acetylcholine, [11C]MK-6884 PET studies in AD patients showed lower cortical binding potential compared to healthy elderly participants, as well as notable cortical heterogeneity and bilateral asymmetry of M4 receptor binding in AD patients. Striatal binding potential was unchanged in these different populations. Our findings demonstrate that the M4 PAM PET tracer [11C]MK-6884 is a useful tool for determining target engagement of M4 PAMs, as well serving as a unique probe to study changes in cholinergic neurotransmission in neurodegenerative disease. The Enigma Biomedical Group has an exclusive license agreement with Merck & Co., Inc., for the global development and commercialization of MK-6884. This allows Enigma and its partner, Cerveau Technologies Inc. to provide biomarkers to industry and academia to accelerate research in the field of neurodegenerative and neuropsychiatric diseases.

Background [ 18 F]MK-6240 is a PET tracer with sub-nanomolar affinity for neurofibrillary tangles. Therefore, tau quantification is possible with [ 18 F]MK-6240 PET/CT scans, and it can be used for assessment of Alzheimer’s disease. However, long acquisition scans are required to provide fully quantitative estimates of pharmacokinetic parameters. Therefore, on the present study, dual-time-window (DTW) acquisitions was simulated to reduce PET/CT acquisition time, while taking into consideration perfusion changes and possible scanning protocol non-compliance. To that end, time activity curves (TACs) representing a 120-min acquisition (TAC 120 ) were simulated using a two-tissue compartment model with metabolite corrected arterial input function from 90-min dynamic [ 18 F]MK-6240 PET scans of three healthy control subjects and five subjects with mild cognitive impairment or Alzheimer’s disease. Therefore, TACs corresponding to different levels of specific binding were generated and then various perfusion changes were simulated. Next, DTW acquisitions were simulated consisting of an acquisition starting at tracer injection, a break and a second acquisition starting at 90 min post-injection. Finally, non-compliance with the PET/CT scanning protocol were simulated to assess its impact on quantification. All TACs were quantified using reference Logan’s distribution volume ratio (DVR) and standardized uptake value ratio (SUVR 90 ) using the cerebellar cortex as reference region. Results It was found that DVR from a DTW protocol with a 60-min break between two 30-min dynamic scans closely approximates the DVR from the uninterrupted TAC 120 , with a regional bias smaller than 2.5%. Moreover, SUVR 90 estimates were more susceptible (regional bias ≤ 19%) to changes in perfusion compared to DVR from a DTW TAC (regional bias ≤ 10%). Similarly, SUVR 90 was affected by late-time scanning protocol delays reaching an increase of 8% for a 20-min delay, while DVR was not affected (regional bias < 1.5%) by DTW protocol non-compliance. Conclusions Therefore, such DTW protocol has the potential to increase patient comfort and throughput without compromising quantitative accuracy and is more reliable against SUVR in terms of perfusion changes and protocol deviations, which could prove beneficial for drug effect assessment and patient follow-up using longitudinal [ 18 F]MK-6240 PET imaging.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

To report on the first group of AD patients studied with a new M4 muscarinic cholinergic receptor PET tracer, [11C]MK-6884.

Positron emission tomography (PET) radioligands (radioactively labelled tracer compounds) are extremely useful for in vivo characterization of central nervous system drug candidates, neurodegenerative diseases and numerous oncology targets 1 . Both tritium and carbon-11 radioisotopologues are generally necessary for in vitro and in vivo characterization of radioligands 2 , yet there exist few radiolabelling protocols for the synthesis of either, inhibiting the development of PET radioligands. The synthesis of such radioligands also needs to be very rapid owing to the short half-life of carbon-11. Here we report a versatile and rapid metallaphotoredox-catalysed method for late-stage installation of both tritium and carbon-11 into the desired compounds via methylation of pharmaceutical precursors bearing aryl and alkyl bromides. Methyl groups are among the most prevalent structural elements found in bioactive molecules, and so this synthetic approach simplifies the discovery of radioligands. To demonstrate the breadth of applicability of this technique, we perform rapid synthesis of 20 tritiated and 10 carbon-11-labelled complex pharmaceuticals and PET radioligands, including a one-step radiosynthesis of the clinically used compounds [ 11 C]UCB-J and [ 11 C]PHNO. We further outline the direct utility of this protocol for preclinical PET imaging and its translation to automated radiosynthesis for routine radiotracer production in human clinical imaging. We also demonstrate this protocol for the installation of other diverse and pharmaceutically useful isotopes, including carbon-14, carbon-13 and deuterium.

PURPOSE:In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES:ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS: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), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS:[18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.

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 ( 89 Zr)-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 89 Zr. 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 (SUV mean ) were quantified by region of interest (ROI) analysis. Results 89 Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89 Zr-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 89 Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89 Zr-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