89 78.41 h) is a positron-emitting radionuclide that displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In the current study, we report the preparation of 89 Zr-desferrioxamine B (DFO)-J591, a novel 89 Zr-labeled monoclonal antibody (mAb) construct for targeted immunoPET and quantification of prostate-specific membrane antigen (PSMA) expression in vivo. Methods: The in vivo behavior of 89 Zr-chloride, 89 Zr-oxalate, and 89 Zr-DFO was studied using PET. High-level computational studies using density functional theory calculations have been used to investigate the electronic structure of 89 Zr-DFO and probe the nature of the complex in aqueous conditions. 89 Zr-DFO-J591 was characterized both in vitro and in vivo. ImmunoPET in male athymic nu/nu mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors was conducted. The change in 89 Zr-DFO-J591 tissue uptake in response to high-and low-specific-activity formulations in the 2 tumor models was measured using acute biodistribution studies and immunoPET. Results: The basic characterization of 3 important reagents-89 Zr-chloride, 89 Zr-oxalate, and the complex 89 Zr-DFO-demonstrated that the nature of the 89 Zr species dramatically affects the biodistribution and pharmacokinetics. Density functional theory calculations provide a rationale for the observed high in vivo stability of 89 Zr-DFO-labeled mAbs and suggest that in aqueous conditions, 89 Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of 2 water molecules. 89 Zr-DFO-J591 was produced in high radiochemical yield (.77%) and purity (.99%), with a specific activity of 181.7 6 1.1 MBq/mg (4.91 6 0.03 mCi/mg). In vitro assays demonstrated that 89 Zr-DFO-J591 had an initial immunoreactive fraction of 0.95 6 0.03 and remained active for up to 7 d. In vivo biodistribution experiments revealed high, target-specific uptake of 89 Zr-DFO-J591 in LNCaP tumors after 24, 48, 96, and 144 h (34.4 6 3.2 percentage injected dose per gram [%ID/g], 38.0 6 6.2 %ID/g, 40.4 6 4.8 %ID/g, and 45.8 6 3.2 %ID/g, respectively). ImmunoPET studies also showed that 89 Zr-DFO-J591 provides excellent image contrast, with tumorto-muscle ratios greater than 20, for the delineation of LNCaP xenografts between 48 and 144 h after administration. Conclusion: These studies demonstrate that 89 Zr-DFO-labeled mAbs show exceptional promise as radiotracers for immunoPET of human cancers. 89 Zr-DFO-J591 displays high tumor-to-background tissue contrast in immunoPET and can be used to delineate and quantify PSMA-positive prostate tumors in vivo.

BackgroundThe positron-emitting radionuclide 89Zr (t 1/2 = 3.17 days) was used to prepare 89Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.Methodology/Principal FindingsTrastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in 89Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. 89Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of 89Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.Conclusions/SignificanceThe results indicate that 89Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.

The topoisomerase-IIα inhibition and antiproliferative activity of α-heterocyclic thiosemicarbazones and their corresponding copper(II) complexes have been investigated. The CuII(thiosemicarbazonato)Cl complexes were shown to catalytically inhibit topoisomerase-IIα at concentrations (0.3–7.2 μM) over an order of magnitude lower than their corresponding thiosemicarbazone ligands alone. The copper complexes were also shown to inhibit the proliferation of breast cancer cells expressing high levels of topoisomerase-IIα (SK-BR-3) at lower concentrations than cells expressing lower levels of the enzyme (MCF-7).

Targeted nanoparticle-based technologies show increasing prevalence in radiotracer design. As a consequence, quantitative contribution of nonspecific accumulation in the target tissue, mainly governed by the enhanced permeability and retention (EPR) effect, becomes highly relevant for evaluating the specificity of these new agents. This study investigated the influence of different tumor phenotypes on the EPR effect, hypothesizing that a baseline level of uptake must be exceeded to visualize high and specific uptake of a targeted macromolecular radiotracer. Methods These preliminary studies use 89Zr-labeled mouse serum albumin (89Zr-desferrioxamine-mAlb) as a model radiotracer to assess uptake and retention in 3 xenograft models of human prostate cancer (CWR22rv1, DU-145, and PC-3). Experiments include PET and contrast-enhanced ultrasound imaging to assess morphology, vascularization, and radiotracer uptake; temporal ex vivo biodistribution studies to quantify radiotracer uptake over time; and histologic and autoradiographic studies to evaluate the intra- and intertumoral distribution of 89Zr-desferrioxamine-mAlb. Results Early uptake profiles show statistically significant but overall small differences in radiotracer uptake between different tumor phenotypes. By 20 h, nonspecific radiotracer uptake was found to be independent of tumor size and phenotype, reaching at least 5.0 percentage injected dose per gram in all 3 tumor models. Conclusion These studies suggest that minimal differences in tumor uptake exist at early time points, dependent on the tumor type. However, these differences equalize over time, reaching around 5.0 percentage injected dose per gram at 20 h after injection. These data provide strong support for the introduction of mandatory experimental controls of future macromolecular or nanoparticle-based drugs, particularly regarding the development of targeted radiotracers.

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes 64Cu and 89Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with 64Cu and 89Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7–5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the 64Cu-DOTA- and 89Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the 64Cu- and 89Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system—one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators—will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.

We evaluated the ability of the PET imaging agent 89Zr-trastuzumab to delineate HER2-positive gastric cancer and to monitor the pharmacodynamic effects of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor afatinib. Methods Using 89Zr-trastuzumab, 18F-FDG, or 3′-deoxy-3′-18F-fluorothymidine (18F-FLT PET), we imaged HER2-positive NCI-N87 and HER2-negative MKN74 gastric cancer xenografts in mice. Next, we examined the pharmacodynamic effects of afatinib in NCI-N87 xenografts using 89Zr-trastuzumab and 18F-FDG PET and comparing imaging results to changes in tumor size and in protein expression as monitored by Western blot and histologic studies. Results Although 18F-FDG uptake in NCI-N87 tumors did not change, a decrease in 89Zr-trastuzumab uptake was observed in the afatinib-treated versus control groups (3.0 ± 0.0 percentage injected dose per gram (%ID/g) vs. 21.0 ± 3.4%ID/g, respectively; P < 0.05). 89Zr-trastuzumab PET results corresponded with tumor reduction, apoptosis, and downregulation of HER2 observed on treatment with afatinib. Downregulation of total HER2, phosphorylated (p)-HER2, and p-EGFR occurred within 24 h of the first dose of afatinib, with a sustained effect over 21 d of treatment. Conclusion Afatinib demonstrated antitumor activity in HER2-positive gastric cancer in vivo. 89Zr-trastuzumab PET specifically delineated HER2-positive gastric cancer and can be used to measure the pharmacodynamic effects of afatinib.

Generally, solid tumors (>400 mm 3 ) are inherently acidic, with more aggressive growth producing greater acidity. If the acidity could be targeted as a biomarker, it would provide a means to gauge the pace of tumor growth and degree of invasiveness, as well as providing a basis for predicting responses to pH-dependent chemotherapies. We have developed a 64 Cu pH (low) insertion peptide (pHLIP) for targeting, imaging, and quantifying acidic tumors by PET, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity, such as hypoxia, lactate dehydrogenase A, and CAIX. We find that the probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.

Introduction-Given the significant utility of suberoylanilide hydroxamic acid (SAHA) in chemotherapeutic protocols, a PET tracer that mimics the histone deacetylase (HDAC) inhibition of SAHA could be a valuable tool in the diagnosis, treatment planning, and treatment monitoring of cancer. Here, we describe the synthesis, characterization, and evaluation of N 1 -(4-(2-[ 18 F]-fluoroethyl)phenyl)-N 8 -hydroxyoctanediamide ([ 18 F]-FESAHA), a PET tracer designed for the delineation of HDAC expression in cancer. Methods-FESAHA was synthesized and biologically characterized in vivo and in vitro.[ 18 F]-FESAHA was then synthesized in high radiochemical purity, and the logP and serum stability of the radiotracer were determined. In vitro cellular uptake experiments and acute biodistribution and small animal PET studies were performed with [ 18 F]-FESAHA in mice bearing LNCaP xenografts. Results-[18 F]-FESAHA was synthesized in high radiochemical purity via an innovative onepot procedure. Enzymatic inhibition assays illustrated that FESAHA is a potent HDAC inhibitor, with IC 50 values from 3 nM to 1.7 μM against the eleven HDAC subtypes. Cell proliferation experiments revealed that the cytostatic properties of FESAHA very closely resemble those of SAHA in both LNCaP cells and PC-3 cells. Acute biodistribution and PET imaging experiments revealed tumor uptake of [ 18 F]-FESAHA and substantially higher values in the small intestine, kidneys, liver, and bone.Conclusion-The significant non-tumor background uptake of [ 18 F]-FESAHA presents a substantial obstacle to the use of the radiotracer as an HDAC expression imaging agent. The study at hand, however, does present a number of lessons critical to both the synthesis of hydroxamic acid containing PET radiotracers and imaging agents aimed at delineating HDAC expression.