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The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin V H /V L regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (K D  = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89 Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for 89Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the 89Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate 89Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new 89Zr-based complex. High stability in vivo, with low accumulation of free 89Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. : We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation technology - was labeled with Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. : The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. : Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

Background Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. Methods The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging. Results The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. Conclusions The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [(11)C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [(11)C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [(11)C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice.

In this study an evaluation of the performance of the Philips MOSAIC small animal PET scanner is presented, with special emphasis on the ability of the system to provide quantitatively accurate PET images. The performance evaluation was structured according to NEMA-like procedures. The transaxial spatial resolution of the system (radial component) ranged between 2.7 mm FWHM at the centre and 3.2 mm FWHM at a radial offset of 45 mm from the centre. The axial spatial resolution of the system ranged between 3.4 mm FWHM at the centre and 5.8 mm FWHM at a radial offset of 45 mm from the centre. The scatter fraction was determined for a mouse- as well as for a rat-sized phantom, and the values obtained were 9.6% and 16.8%, respectively. For the mouse phantom, the maximum count rate measured was 560 kcps at 93 MBq; the maximum NEC rate equalled 308 kcps at 1.7 MBq/ml. For the rat phantom, these values were 400 kcps at 100 MBq and 129 kcps at 0.24 MBq/ml, respectively. The sensitivity of the system was derived to be 0.65%. An energy window between 410 and 665 keV was used in all experiments. The MOSAIC system exhibits moderate spatial resolution and sensitivity values, but good NEC performance. In combination with its relatively large field of view, the system allows for high-throughput whole-body imaging of mice and rats. The accurate measurement of relative changes in radiotracer distributions is feasible.

The novel PET flow tracer flurpiridaz F 18 shows high myocardial extraction and slow washout. flurpiridaz F 18 PET data analysis with tracer kinetic modeling provides accurate absolute myocardial blood flow (MBF) measurements but requires in-scanner injection and complex processing. We evaluated the hypothesis that myocardial retention and standardized uptake values (SUVs) based on late uptake provide accurate estimates of myocardial flow reserve (MFR) and, thus, might allow simplified quantification after tracer injection outside the scanner. Nine pigs had dynamic PET scans after repeated injections of flurpiridaz F 18 at rest and combined adenosine and dobutamine stress. flurpiridaz F 18 PET with a 3-compartment model and coinjected radioactive microspheres were used to delineate MBF. These quantitative measurements were compared with myocardial retention (%/min) and SUV of flurpiridaz F 18 after summing data over 5-10, 5-12, 5-15, 10-15, and 10-20 min after tracer injection. MBF ranged from 0.5 to 2.8 mL/min/g. There was a good correlation between both flurpiridaz F 18 retention and SUVs from 5 to 12 min after injection and MBF measured using 3-compartment model- or microsphere-derived MBF (r = 0.73, P < 0.05, and r = 0.68, P < 0.05, respectively, for retention; r = 0.88, P < 0.001, and r = 0.92, P < 0.001, respectively, for SUV). At later time points, retention and SUV underestimated stress microsphere flow (at 10-20 min: r = 0.41, P = not significant, and r = 0.46, P = not significant, respectively, for retention; r = 0.41, P = not significant, and r = 0.65, P < 0.05, respectively, for SUV). When measured 5-12 min after injection, there was a close agreement between MFR measured with either flurpiridaz F 18 retention or SUV and MFR measured using microspheres (mean difference, -0.08 ± 0.36 and -0.18 ± 0.25, respectively). Myocardial retention and SUVs of the (18)F-labeled flow tracer flurpiridaz F 18 accurately reflect the MFR. These simplified analysis methods may facilitate the combination of quantitative assessment of perfusion reserve and rapid clinical imaging protocols.

Conventional myocardial perfusion PET tracers require onsite tracer production because of their short radioactive half-lives. To investigate the potential of a new (18)F-labeled pyridazinone analog ((18)F-BMS-747158-02), we characterized this tracer in a rat model of permanent and transient coronary occlusion using small-animal PET. Myocardial (18)F-BMS-747158-02 distribution in healthy rats (n = 7), rats with transient (3-min) left coronary artery occlusion (n = 11), and rats with permanent left coronary occlusion (n = 11) was analyzed with a dedicated small-animal PET scanner. Normal hearts demonstrated intense and almost homogeneous tracer uptake throughout the left ventricle for more than 2 h. During permanent coronary occlusion, PET demonstrated perfusion defects, which remained unchanged (37.6% +/- 8.8%, 37.4% +/- 10.2%, and 36.2% +/- 9.8% left ventricle at 15, 45, and 115 min, respectively, after tracer injection). After transient ischemia, the induced defect size decreased significantly after reperfusion (16.2% +/- 9.3%, 6.0% +/- 6.5%, and 1.4% +/- 1.3% left ventricle). Tracer reinjection after transient ischemia resulted in normalization of the induced defect. Coronary occlusion yielded distinct myocardial (18)F-BMS-747158-02 uptake defects in the area of ischemia, which demonstrated normalization of activity after reperfusion and reinjection. These promising kinetic parameters may allow for assessment of flow using exercise-rest protocols similar to those used in combination with exercise and rest perfusion SPECT.

18F-galacto-RGD (18F-RGD) is a PET tracer binding to alpha v beta 3 integrin receptors that are upregulated after myocardial infarction (MI) as part of the healing process. We studied whether myocardial 18F-RGD uptake early after MI is associated with long-term left-ventricle (LV) remodeling in a rat model. METHODS: Wistar rats underwent sham operation (n = 9) or permanent coronary ligation (n = 25). One week after MI, rats were injected with 18F-RGD to evaluate alpha v beta 3 integrin expression using a preclinical PET system. In the same rats, LV volumes and defect size were measured 1 and 12 wk after MI by 13N-ammonia PET and MRI, respectively. RESULTS: One week after MI, 18F-RGD uptake was increased in the defect area as compared with the remote myocardium of MI rats or sham-operated controls (percentage injected dose per cubic centimeter, 0.20 plus or minus 0.05 vs. 0.06 plus or minus 0.03 and 0.07 plus or minus 0.04, P < 0.001). At this time, 18F-RGD uptake was associated with capillary density in histologic sections. Average 18F-RGD uptake in the defect area was lowest in the rats demonstrating greater than 20% relative increase in the LV end-diastolic volume from 1 to 12 wk (percentage injected dose per centimeter cubed, 0.15 plus or minus 0.07 vs. 0.21 plus or minus 0.05, P < 0.05). In a multivariable logistic regression analysis, low 18F-RGD uptake was a significant predictor of increase in end-diastolic volume (r = 0.51, P < 0.05). CONCLUSION: High levels of 18F-RGD uptake in the perfusion defect area early after MI were associated with the absence of significant LV remodeling after 12 wk of follow-up. These results suggest that alpha v beta 3 integrin expression is a potential biomarker of myocardial repair processes after MI and enables the monitoring of these processes by molecular imaging to derive possible prognostic information.

PET allows for quantitative, regional myocardial perfusion imaging. The short half-lives of the perfusion tracers currently in use limit their clinical applicability. Here, the biodistribution and imaging quality of a new 18F-labeled myocardial perfusion agent (18F-BMS-747158-02) in an animal model are described. The biodistribution of 18F-BMS-747158-02 was determined at 10 and 60 min after injection. The first-pass extraction fraction of the tracer was measured in isolated rat hearts perfused with the Langendorff method. Small-animal PET imaging was used to study tracer retention. The biodistribution at 10 min after injection demonstrated high myocardial uptake (3.1 percentage injected dose per gram [%ID/g]) accompanied by little activity in the lungs (0.3 %ID/g) and liver (1.0 %ID/g). The tracer showed a high and flow-independent myocardial first-pass extraction fraction, averaging 0.94 (SD = 0.04). PET imaging provided excellent delineation of myocardial structures. The heart-to-lung activity ratio increased from 4.7 to 10.2 between 1 and 15 min after tracer injection (at rest). Adenosine infusion (140 microg/kg/min) led to a significant increase in myocardial tracer retention (from 1.68 [SD = 0.23]) s(-1) to 3.21 [SD = 0.92] s(-1); P = 0.03). The observation of a high and flow-independent first-pass extraction fraction promises linearity between tracer uptake and myocardial blood flow. Sustained myocardial tracer uptake, combined with high image contrast, will allow for imaging protocols with tracer injection at peak exercise followed by delayed imaging. Thus, 18F-BMS-747158-02 is a promising new tracer for the quantitative imaging of myocardial perfusion and can be distributed to imaging laboratories without a cyclotron.