Lars R. Perk

In Vivo VEGF Imaging with Radiolabeled Bevacizumab in a Human Ovarian Tumor Xenograft

Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for noninvasive in vivo VEGF visualization and quantification with the single γ-emitting isotope 111In and the PET isotope 89Zr. Methods: Labeling, stability, and binding studies were performed. Nude mice with a human SKOV-3 ovarian tumor xenograft were injected with 89Zr-bevacizumab, 111In-bevacizumab, or human 89Zr-IgG. Human 89Zr-IgG served as an aspecific control antibody. Small-animal PET and microCT studies were obtained at 24, 72, and 168 h after injection of 89Zr-bevacizumab and 89Zr-IgG (3.5 ± 0.5 MBq, 100 ± 6 μg, 0.2 mL [mean ± SD]). Small-animal PET and microCT images were fused to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. 89Zr- and 111In-bevacizumab ex vivo biodistribution was compared at 24, 72, and 168 h after injection (2.0 ± 0.5 MBq each, 100 ± 4 μg in total, 0.2 mL). Results: Labeling efficiencies, radiochemical purity, stability, and binding properties were optimal for the radioimmunoconjugates. Small-animal PET showed uptake in well-perfused organs at 24 h and clear tumor localization from 72 h onward. Tumor uptake determined by quantification of small-animal PET images was higher for 89Zr-bevacizumab—namely, 7.38 ± 2.06 %ID/g compared with 3.39 ± 1.16 %ID/g (percentage injected dose per gram) for human 89Zr-IgG (P = 0.011) at 168 h and equivalent to ex vivo biodistribution studies. Tracer uptake in other organs was seen primarily in liver and spleen. 89Zr- and 111In-bevacizumab biodistribution was comparable. Conclusion: Radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Noninvasive quantitative small-animal PET was similar to invasive ex vivo biodistribution. Radiolabeled bevacizumab is a new tracer for noninvasive in vivo imaging of VEGF in the tumor microenvironment.

Development and Characterization of Clinical-Grade 89Zr-Trastuzumab for HER2/neu ImmunoPET Imaging

The anti–human epidermal growth factor receptor 2 (HER2/neu) antibody trastuzumab is administered to patients with HER2/neu-overexpressing breast cancer. Whole-body noninvasive HER2/neu scintigraphy could help to assess and quantify the HER2/neu expression of all lesions, including nonaccessible metastases. The aims of this study were to develop clinical-grade radiolabeled trastuzumab for clinical HER2/neu immunoPET scintigraphy, to improve diagnostic imaging, to guide antibody-based therapy, and to support early antibody development. The PET radiopharmaceutical 89Zr-trastuzumab was compared with the SPECT tracer 111In-trastuzumab, which we have tested in the clinic already. Methods: Trastuzumab was labeled with 89Zr and (for comparison) with 111In. The minimal dose of trastuzumab required for optimal small-animal PET imaging and biodistribution was determined with human HER2/neu-positive or -negative tumor xenograft–bearing mice. Results: Trastuzumab was efficiently radiolabeled with 89Zr at a high radiochemical purity and specific activity. The antigen-binding capacity was preserved, and the radiopharmaceutical proved to be stable for up to 7 d in solvent and human serum. Of the tested protein doses, the minimal dose of trastuzumab (100 μg) proved to be optimal for imaging. The comparative biodistribution study showed a higher level of 89Zr-trastuzumab in HER2/neu-positive tumors than in HER2/neu-negative tumors, especially at day 6 (33.4 ± 7.6 [mean ± SEM] vs. 7.1 ± 0.7 percentage injected dose per gram of tissue). There were good correlations between the small-animal PET images and the biodistribution data and between 89Zr-trastuzumab and 111In-trastuzumab uptake in tumors (R2 = 0.972). Conclusion: Clinical-grade 89Zr-trastuzumab showed high and HER2/neu-specific tumor uptake at a good resolution.

Immuno-PET: A Navigator in Monoclonal Antibody Development and Applications

Monoclonal antibodies (mAbs) have been approved for use as diagnostics and therapeutics in a broad range of medical indications, but especially in oncology. In addition, hundreds of new mAbs, engineered mAb fragments, and nontraditional antibody-like scaffolds directed against either validated or novel tumor targets are under development. Immuno-positron emission tomography (PET), the tracking and quantification of mAbs with PET in vivo, is an exciting novel option to improve diagnostic imaging and to guide mAb-based therapy. In this review, recent technical advances leading to a jump ahead in mAb imaging are discussed. The availability of proper positron emitters, sophisticated radiochemistry, and advanced PET and PET-computed tomography scanners is crucial in these developments. Immuno-PET might play an important future role in cancer staging, in the improvement and tailoring of therapy with existing mAbs, and in the efficient development of novel mAbs. An overview of the preclinical and first clinical immuno-PET studies is provided.

Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine.

The positron emitter zirconium-89 (89Zr) has very attractive properties for positron emission tomography (PET) imaging of intact monoclonal antibodies (mAbs) using immuno-PET. This protocol describes the step-by-step procedure for the facile radiolabeling of mAbs or other proteins with 89Zr using p-isothiocyanatobenzyl-desferrioxamine (Df–Bz–NCS). First, Df–Bz–NCS is coupled to the lysine–NH2 groups of a mAb at pH 9.0 (pre-modification), followed by purification using gel filtration. Next, the pre-modified mAb is labeled at room temperature by the addition of [89Zr]Zr-oxalic acid solution followed by purification using gel filtration. The entire process of pre-modification, radiolabeling and purification steps will take about 2.5 h.

Synthesis and initial preclinical evaluation of the P2X7 receptor antagonist [11C]A-740003 as a novel tracer of neuroinflammation

Neuroinflammation, in particular activation of microglia, is thought to play an important role in the progression of neurodegenerative diseases. In activated microglia, the purinergic P2X7 receptor is upregulated. A-740003, a highly affine and selective P2X7 receptor antagonist, is a promising candidate for the development of a radiotracer for imaging of neuroinflammation by positron emission tomography. For this purpose, [(11)C]A-740003 was synthesised and evaluated in vivo with respect to both tracer metabolism and biodistribution. In plasma, a moderate metabolic rate was seen. In healthy rat brain, only marginal uptake of [(11)C]A-740003 was observed and, therefore, metabolites in brain could not be determined. Whether the minimal brain uptake is due to the low expression levels of the P2X7 receptor in healthy brain or to limited transport across the blood-brain barrier has yet to be elucidated.

11C‐labeled and 18F‐labeled PET ligands for subtype‐specific imaging of histamine receptors in the brain

The signaling molecule histamine plays a key role in the mediation of immune reactions, in gastric secretion, and in the sensory system. In addition, it has an important function as a neurotransmitter in the central nervous system, acting in pituitary hormone secretion, wakefulness, motor and cognitive functions, as well as in itch and nociception. This has raised interest in the role of the histaminergic system for the treatment and diagnosis of various pathologies such as allergy, sleeping and eating disorders, neurodegeneration, neuroinflammation, mood disorders, and pruritus. In the past 20 years, several ligands targeting the four different histamine receptor subtypes have been explored as potential radiotracers for positron emission tomography (PET). This contribution provides an overview of the developments of subtype-selective carbon-11-labeled and fluorine-18-labeled compounds for imaging in the brain. Using specific radioligands, the H1 R expression in human brain could be examined in diseases such as schizophrenia, depression, and anorexia nervosa. In addition, the sedative effects of antihistamines could be investigated in terms of H1 R occupancy. The H3 R is of special interest because of its regulatory role in the release of various other neurotransmitters, and initial H3 R PET imaging studies in humans have been reported. The H4 R is the youngest member of the histamine receptor family and is involved in neuroinflammation and various sensory pathways. To date, two H4 R-specific (11) C-labeled ligands have been synthesized, and the imaging of the H4 R in vivo is in the early stage.

High resolution PET imaging characteristics of /sup 68/Ga, /sup 124/I and /sup 89/Zr compared to /sup 18/F

In this study the imaging characteristics influencing the quantitative accuracy of 68Ga, 124I and 89 Zr were determined and compared to those of 18F using a 3D high resolution PET (high resolution research tomograph, HRRT) scanner. Although there were large discrepancies found in the sensitivity of these isotopes, which can be explained by their positron abundancy, none of the assessed imaging characteristic prevents the isotopes for usage in (high resolution) quantitative PET imaging. Care has to be taken, however, that accurate correction methods are used for dead time, background and scatter, and partial volume

Technical Note: Building a combined cyclotron and MRI facility: Implications for interference

PURPOSE With the introduction of hybrid PET∕MRI systems, it has become more likely that the cyclotron and MRI systems will be located close to each other. This study considered the interference between a cyclotron and a superconducting MRI system. METHODS Interactions between cyclotrons and MRIs are theoretically considered. The main interference is expected to be the perturbation of the magnetic field in the MRI due to switching on or off the magnetic field of the cyclotron. MR imaging is distorted by a dynamic spatial gradient of an external inplane magnetic field larger than 0.5-0.04 μT∕m, depending on the specific MR application. From the design of a cyclotron, it is expected that the magnetic fringe field at large distances behaves as a magnetic dipolar field. This allows estimation of the full dipolar field and its spatial gradients from a single measurement. Around an 18 MeV cyclotron (Cyclone, IBA), magnetic field measurements were performed on 5 locations and compared with calculations based upon a dipolar field model. RESULTS At the measurement locations the estimated and measured values of the magnetic field component and its spatial gradients of the inplane component were compared, and found to agree within a factor 1.1 for the magnetic field and within a factor of 1.5 for the spatial gradients of the field. In the specific case of the 18 MeV cyclotron with a vertical magnetic field and a 3T superconducting whole body MR system, a minimum distance of 20 m has to be considered to prevent interference. CONCLUSIONS This study showed that a dipole model is sufficiently accurate to predict the interference of a cyclotron on a MRI scanner, for site planning purposes. The cyclotron and a whole body MRI system considered in this study need to be placed more than 20 m apart, or magnetic shielding should be utilized.

Radioimmunotherapy for Indolent B-Cell Non-Hodgkin Lymphoma in Relapsed, Refractory and Transformed Disease

Radioimmunotherapy (RIT) is a new treatment modality that combines the benefits of radiotherapy and immunotherapy. In RIT, a radionuclide is coupled to a monoclonal antibody, directed against an antigen expressed on tumor cells. Recently, RIT has been introduced targeting the CD20 surface antigen, which is expressed on nearly all B-cell non-Hodgkin lymphomas (NHL).Clinical experience with RIT in the treatment of patients with indolent NHL is increasing. To date, two commercially available agents are used: yttrium-90 (90Y)-ibritumomab tiuxetan and iodine-131 (131I)-tositumomab. In general, there is no organ-specific non-hematologic toxicity when a standard dose of RIT is used. Bone marrow suppression is the dose-limiting RIT toxicity; therefore, bone marrow infiltration by NHL should be investigated before treatment. Treatment-related myelodysplastic syndromes and acute myeloid leukemia after RIT are being investigated but long-term data are needed for final evaluation.Results are quite encouraging with respect to complete remission and overall response, even in pretreated patients with unconjugated monoclonal antibodies. RIT induces high response rates and a significant subgroup of patients has achieved long-term durable responses. RIT is feasible in heavily pretreated patients and does not compromise future treatments in the event of progressive disease. Randomized phase III studies are in progress to evaluate the timing of RIT in the overall management of indolent NHLInvestigations of new emerging therapeutic strategies for patients with indolent NHL are underway, with research into the feasibility of RIT as first-line therapy and in advanced disease, RIT dose escalation and combined modality approaches with autologous stem cell transplantation. The encouraging results of RIT in indolent NHL have initiated studies focusing on its benefit for patients with aggressive NHL.

Imaging Histamine Receptors Using PET and SPECT

The histaminergic system contains four subtypes of G-protein-coupled receptors: H1R, H2R, H3R and H4R. In the central nervous system, they mediate the action of histamine in pituitary hormone secretion, wakefulness, motor and cognitive functions as well as in itch and nociception. Alterations in the cerebral histaminergic neurotransmission are associated with numerous neurological diseases, e.g. sleep/wake and eating disorders, epilepsy, neuropathic pain, neurodegeneration and neuroinflammation. Therefore, histamine receptors are a target of high interest, not only for therapy, but also for imaging and quantification using PET and SPECT. In addition to indirect flow and metabolism studies, this chapter provides a review of results of preclinical and clinical investigations with subtype-selective tracers.