Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant ß1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.

The Positron Emission Tomography Tracer 3'-Deoxy-3'-[18F]Fluorothymidine ([18F]FLT) Is Not Suitable to Detect Tissue Proliferation Induced by Systemic Yersinia enterocolitica Infection in Mice.

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis.

Preclinical evaluation of the anti-tumor effects of the natural isoflavone genistein in two xenograft mouse models monitored by [18F]FDG, [18F]FLT, and [64Cu]NODAGA-cetuximab small animal PET.

UNLABELLED: The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. PROCEDURES: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [18F]FDG, [18F]FLT and [64Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. RESULTS: Genistein reduced tumor growth significantly in both xenografts. [18F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [18F]FDG and [64Cu]NODAGA-cetuximab were not suitable for therapy monitoring. CONCLUSIONS: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [18F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

New pathogen-specific immunoPET/MR tracer for molecular imaging of a systemic bacterial infection.

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Evaluation of the Metabolic Activity of Echinococcus multilocularis in Rodents Using Positron Emission Tomography Tracers.

PURPOSE: 2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) has been used as a standard clinical positron emission tomography (PET) tracer for the follow-up of the rare but life-threatening parasitic disease alveolar echinococcosis (AE). Given that the disease is endemic in many countries in the northern hemisphere and the diagnosis is still challenging, the aim of our study was to evaluate further clinically relevant PET tracers as possible diagnostic tools for AE in vitro and in vivo. PROCEDURES: Various clinically used PET tracers were evaluated in vitro and assessed in an in vivo AE animal model based on PET/magnetic resonance (MR) measurements. RESULTS: In vitro binding assays displayed high uptake of [(18)F]FDG in a cell suspension of E. multilocularis tissue, whereas 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) and [(11)C]choline were found to be taken up strongly by E. multilocularis vesicles. [(18)F]FDG and [(18)F]FLT displayed an elevated uptake in vivo, which appeared as several foci throughout the parasite tissue as opposed to [(18)F]fluoro-azomycinarabinofuranoside ([(18)F]FAZA) and [(11)C]choline. CONCLUSIONS: Our data clearly demonstrate that the clinically applied PET tracer [(18)F]FDG is useful for the diagnosis and disease staging of AE but also has drawbacks in the assessment of currently inactive or metabolically weak parasitic lesions. The different tested PET tracers do not show the potential for the replacement or supplementation of current diagnostic strategies. Hence, there is still the need for novel diagnostic tools.

The impact of weakly bound 89Zr on preclinical studies: non-specific accumulation in solid tumors and aspergillus infection.

UNLABELLED: Preclinical studies involving (89)Zr often report significant bone accumulation, which is associated with dissociation of the radiometal from the tracer. However, experiments determining the uptake of unbound (89)Zr in disease models are not performed as routine controls. The purpose of the present study was to investigate the impact of free or weakly bound (89)Zr on PET quantifications in disease models, in order to determine if such control experiments are warranted. METHODS: Chemical studies were carried out to find a (89)Zr compound that would solubilize the (89)Zr as a weak chelate, thus mimicking free or weakly bound (89)Zr released in circulation. (89)Zr oxalate had the desired characteristics, and was injected into mice bearing FaDu and HT29 solid tumor xenografts, and mice infected in the lungs with the mold Aspergillus fumigatus, as well as in healthy controls (naïve). PET/CT or PET/MR imaging followed to quantify the distribution of the radionuclide in the disease models. RESULTS: (89)Zr oxalate was found to have a plasma half-life of 5.1 ± 2.3 h, accumulating mainly in the bones of all animals. Both tumor types accumulated (89)Zr on the order of 2-4 %ID/cm(3), which is comparable to EPR-mediated accumulation of certain species. In the aspergillosis model, the concentration of (89)Zr in lung tissue of the naïve animals was 6.0 ± 1.1 %ID/g. This was significantly different from that of the animals with advanced disease, showing 11.6 ± 1.8 %ID/g. CONCLUSIONS: Given the high levels of (89)Zr accumulation in the disease sites in the present study, we recommend control experiments mapping the biodistribution of free (89)Zr in any preclinical study employing (89)Zr where bone uptake is observed. Aqueous (89)Zr oxalate appears to be a suitable compound for such studies. This is especially relevant in studies where the tracer accumulation is based upon passive targeting, such as EPR.

Preclinical and Translational PET/MR Imaging.

Combined PET and MR imaging (PET/MR imaging) has progressed tremendously in recent years. The focus of current research has shifted from technologic challenges to the application of this new multimodal imaging technology in the areas of oncology, cardiology, neurology, and infectious diseases. This article reviews studies in preclinical and clinical translation. The common theme of these initial results is the complementary nature of combined PET/MR imaging that often provides additional insights into biologic systems that were not clearly feasible with just one modality alone. However, in vivo findings require ex vivo validation. Combined PET/MR imaging also triggers a multitude of new developments in image analysis that are aimed at merging and using multimodal information that ranges from better tumor characterization to analysis of metabolic brain networks. The combination of connectomics information that maps brain networks derived from multiparametric MR data with metabolic information from PET can even lead to the formation of a new research field that we would call cometomics that would map functional and metabolic brain networks. These new methodologic developments also call for more multidisciplinarity in the field of molecular imaging, in which close interaction and training among clinicians and a variety of scientists is needed.

Pharmacokinetics and PET imaging properties of two recombinant anti-PSMA antibody fragments in comparison to their parental antibody.

BACKGROUND: Radioimmunoimaging with disease-specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno-positron emission tomography (PET). In this study, PSMA-specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts. METHODS: Recombinant anti-PSMA antibody fragments D7-Fc and D7-CH3 were constructed by genetically fusing the binding domains of mAb 3/F11 (D7) to the human IgG3 CH3 or CH2-CH3 (Fc) domain. The fragments and the mAb 3/F11 were DOTA conjugated, tested in vitro, and radiolabeled with (64) Cu. PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used for PET-MR imaging and for ex vivo biodistribution. RESULTS: The constructs showed strong and specific binding to PSMA-positive C4-2 cells in vitro which did not decrease after DOTA conjugation. Both tested fragments showed stable accumulation in PSMA-positive C4-2 tumors at all measured time points but reduced uptake compared to the full-length antibody. Other organs and PSMA-negative tumors showed a very low tracer uptake only 3 hr after injection, with the exception of the kidneys, which demonstrated high radioactivity uptake due to rapid renal clearance of the mAb fragments. CONCLUSION: Stable tumor uptake and fast serum clearance of the tested radiolabeled fragments was observed in this preclinical study compared to the full length mAb. Since the fragments show rapid and specific tumor uptake, the tested fragments might serve as tools for theranostic imaging with suitable isotopes for radioimmunotherapy.

Methodologic issues in the measurement of interleukin-16 in clinical blood samples using immunoassays.

Quantitation of interleukin-16 (IL-16) in clinical blood samples has strongly increased, since IL-16 appears to be involved in the pathogenesis of several inflammatory diseases. IL-16 is synthesized in the cell cytoplasm as precursor protein (pro-IL-16), which can be processed by caspase-3 into N-terminal (N-IL-16) and C-terminal (C-IL-16) fragments. C-IL-16 is described to be subsequently secreted. Using commercially available IL-16 ELISA, a pro-IL-16 ELISA and immunoprecipitation analysis, we investigated, whether type and handling of blood samples influence IL-16 quantitation and whether existing IL-16 ELISA are specific for C-IL-16. We observed that cell-rich plasma samples reflect falsely-elevated IL-16 concentrations due to cell contaminations. Interestingly, not C-IL-16, but pro-IL-16 represents the major IL-16 form in cell-rich plasma samples. Notably, commercially IL-16 ELISA could not distinguish between C-IL-16 and pro-IL-16. Thus, cell-rich plasma samples should not be used for IL-16 measurements and new methods are necessary for quantitation of C-IL-16 and pro-IL-16 uniquely.