Systematically Evaluating DOTATATE and FDG as PET Immuno-Imaging Tracers of Cardiovascular Inflammation

The somatostatin receptor 2-binding PET tracer DOTATATE is emerging as an alternative to 18F-FDG to assess cardiovascular inflammation. The strengths and weaknesses of each tracer and their different specificity for inflammatory cells still need to be fully elucidated. In this manuscript, we employed mouse and rabbit animal models of inflammation. In mice, 64Cu-DOTATATE’s pharmacokinetics and timed biodistribution were determined in control (C57BL/6) and atherosclerotic (Apoe−/−) mice by ex vivo gamma counting. In vivo PET/CT, combined with ex vivo flow cytometry and gamma counting, was used to evaluate the quantification of cardiovascular inflammation by 64Cu-DOTATATE and 18F-FDG and the tracers’ cellular specificity in control versus infarcted and atherosclerotic mice. In a translational PET/MRI rabbit study, we then compared DOTATATE labeled with short-lived radioisotope 68Ga and 18F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. In infarcted mice, in vivo 64Cu-DOTATATE PET showed higher differential uptake than 18F-FDG between infarcted and remote myocardium (p=0.0286), and with respect to controls (p=0.0286; n=4-6). In atherosclerotic mice, 64Cu-DOTATATE PET aortic signal, but not 18F-FDG, was higher compared to controls (p=0.0286; n=4). In both models, 64Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while 18F-FDG uptake was less cell-specific. The translational rabbit PET/MRI study showed significantly higher aortic accumulation of both 68Ga-DOTATATE and 18F-FDG in atherosclerotic compared to control animals (p=0.0002 and p=0.0159, respectively; n=10-32). In conclusion, we introduce a workflow integrating in vivo PET and ex vivo immunological and radioactivity counting assays to characterize DOTATATE and 18F-FDG as inflammation tracers in small animal models of cardiovascular disease. Our results support the use of DOTATATE to assess cardiovascular inflammation, as alternative and complement to 18F-FDG. In addition, our study establishes a comprehensive and robust framework for the thorough assessment and comparison of novel and validated PET immuno-tracers in the cardiovascular arena.

Nasal oxytocin for the treatment of psychiatric disorders and pain: achieving meaningful brain concentrations

There is evidence of the therapeutic potential of intranasal oxytocin for the treatment of pain and various psychiatric disorders, however, there is scant evidence that oxytocin reaches the brain. We quantified the concentration and distribution pattern of [125I]-radiolabeled oxytocin in the brains and peripheral tissues of rats after intranasal delivery using gamma counting and autoradiography, respectively. Radiolabel was detected in high concentrations in the trigeminal and olfactory nerves as well as in brain regions along their trajectories. Considerable concentrations were observed in the blood, however, relatively low levels of radiolabel were measured in peripheral tissues. The addition of a mucoadhesive did not enhance brain concentrations. These results provide support for intranasal OT reaching the brain via the olfactory and trigeminal neural pathways. These findings will inform the design and interpretation of clinical studies with intranasal oxytocin.

138 In vivo localization of genetically engineered natural killer cells against glioblastoma using PET imaging

BackgroundGlioblastoma (GBM) is a deadly brain malignancy with a dismal prognosis. While immunotherapy holds great promise for GBM treatment, most have failed due to a suppressive tumor microenvironment (TME). Antigen heterogeneity and adenosine signaling are two immunosuppressive mechanisms in GBM. The CD73-adenosine axis plays a multifaceted role in GBM pathogenesis and drives the dysfunction of NK cells in GBM TME.1,3 Our NKG2D-chimeric antigen receptor (CAR)-natural killer (NK) cells have shown anti-tumor activity when combined with CD73 blockade in vivo.2 To further extend the potency of these cells against GBM and address antigen heterogeneity in GBM, we combined the local blockade of CD73 with multi-antigen-targeting engineered NK cells. In order to improve treatment assessment, PET/MR imaging was employed to enable detailed, non-invasive assessment of tumor progression. Imaging assessment of adoptively-transferred CAR- NK cells was also developed to determine the fate of NK cell delivery to the tumor site over time.MethodsWe generated multifunctional engineered NK (E-NK) cells that express an anti-CD73 scFv, which is cleavable by GBM-associated proteases, an NKG2D-CAR, as well as a GD2 CAR, which can actively target the GD2 antigen overexpressed on GBM (Figure 1A). For E-NK cell radiolabeling, zirconium-89 (89Zr, ½ life = 78 Hr) radiotracer was attached covalently to the E-NK cell surface via conjugation with DFO-Bz-NCS in a range of doses from 50–600 µCi.ResultsAn optimal balance between labeling efficiency and cell viability was attained at 120 µCi 89Zr resulting in 39% labeling efficiency and 46% cell viability over for 48 hours. After labeling, the NK cells maintained their in vitro killing activity against GBM cells (figure 1B). The 89Zr labeled E-NK cells were administered intravenously in mice containing intracranial GBM10 tumors at week 5 post-implant. PET imaging was performed at 1 and 2 days later and gamma imaging ex vivo at 4 days. Free 89Zr was visible diffusely throughout the body with low levels in the brain. The majority of 89Zr labeled E-NK cell groups localized to the lungs with detectable activity elsewhere in various organs (figure 1C and 1D).Abstract 138 Figure 1PET imaging and gamma counting of the engineered NK cellsFigure 1 (A) Multifunctional, responsive CAR constructs; (B) In vitro killing activity against GBM43 cells after co-incubation with 89Zr labeled NK cells at an E:T ratio of 10 for 4 h with LDH assay (N=3); (C) & (D) In vivo PET imaging and ex vivo gamma counting with 89Zr at week 5 in 10 mice during 4 days, GBM intracranial implantation to NSG male mouse, 89Zr, 89Zr + NK cell, or 89Zr + E NK cell (7 × 106 cells with 500 µCi) was administered through intravenous injection, Qimage was used for the PET/MRI co-registration and analysisConclusionsWe generated multifunctional E-NK cells which showed the improved killing of GBM cells using novel targeting approaches, including the blockade of CD73-mediated adenosinergic signaling. We also optimized E-NK cell radiolabeling with 89Zr for GB10 therapy in vitro and in vivo fate mapping against a xenograft of patient-derived GBM.AcknowledgementsWe gratefully acknowledge the Walther Oncology Embedding Program, Indiana University Simon Cancer Center, and In Vivo Therapeutics Core.ReferencesWang J, Matosevic S. NT5E/CD73 as correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8(10):1526.Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6(1):136.Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci 2019;39(22):4387.Flink J, Muzi M, Peck M, Krohn K. Multimodality brain tumor imaging: mr imaging, PET, and PET/MR imaging. J Nucl 2015;5(10):1554–1561.

Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis

Background B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE). Methods Female C57BL/6 J mice were induced with EAE through immunization with myelin oligodendrocyte glycoprotein (MOG1–125). PET imaging of naïve and EAE mice was performed 19 h after administration of [64Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted. Results Radiolabelling of DOTA-conjugated CD19-mAb with 64Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/μmol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02 ± 0.092 vs. 1.68 ± 0.06 percentage of injected dose per gram, % ID/g, p = 0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22 ± 0.020 vs. 0.12 ± 0.003 % ID/g, p = 0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry—providing further evidence that [64Cu]CD19-mAb enables visualization of B cell infiltration into the CNS of EAE mice. Conclusion CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis

Background: B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE).Methods: Female C57BL/6J mice were induced with EAE through immunisation with myelin oligodendrocyte glycoprotein (MOG1–125). PET imaging of naïve and EAE mice was performed 19 h after administration of [64Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted.Results: Radiolabelling of DOTA-conjugated CD19-mAb with 64Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/mol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02±0.092 vs. 1.68±0.06 percentage of injected dose per gram, %ID/g, p=0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22±0.020 vs. 0.12±0.003 %ID/g, p=0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry – providing further evidence that [64Cu]CD19-mAb enables visualisation of B cell infiltration into the CNS of EAE mice. Conclusion: CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm

Background Abdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). Methods and Results Fourteen-week-old apolipoprotein E-knockout (ApoE−/−) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001). Conclusions [18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents

PEG coated magnetic nanocarriers avoid the reticuloendothelial system, and show an MRI contrast in the kidneys. The results are supported by SPECT, gamma-counting, MRI and TEM histology.