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PDF file - 165K, Additional SPECT/CT images of the uPAR probes imaging the MDA-MB-231 CDM

Components of the plasminogen activation system, which are overexpressed in aggressive breast cancer subtypes, offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared imaging and 111In single-photon emission computed tomography. Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard 18F-fluorodeoxyglucose at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. In addition, a radioimmunotherapy study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope 177Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof of concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology. Cancer Res; 73(7); 2070–81. ©2013 AACR.

PDF file - 38K, Determining the half-life of the 111In-labeled antibodies in vivo (n = 3 mice / antibody)

PDF file - 62K, FDG avid lesion not detected by 111In-2G10

PDF file - 62K

Supplemental Figures S1-S6. U33 Fab Sequence (S1); Inhibition constant calculation of U33 Fab (S2); U33 does not bind to the uPA-PAI1 complex (S3); Selectivity of U33 IgG for active uPA versus inactive uPA (S4); Dependence of U33 IgG binding on an accessible uPA active site (S5); Selectivity of U33 IgG for human uPA compared to highly related serine proteases (S6).

PDF file - 41K, The affinity of 2G10 and 3C6 Fabs and IgGs for MDA-MB-231 cells was determined by equilibrium binding flow cytometry. Isotherms were fit using non-linear regression methods to calculate KDs

PDF file - 101K, Mouse model recreating the inflammation observed in Figure 4C. A nude mouse was injected subcutaneously with 100�l of turpentine oil. After 72hrs, the animal was injected with 258°Ci of FDG and imaged 60 minutes later. Probe uptake by the inflammatory cells is only observed with FDG and not with the uPAR probe

PDF file - 71K, Scrambled matched isotype control IgGs labeld with AlexaFlupr 680 at 72hrs in MDA-MB-231 xenografts imaged in the Cy 5.5 channel at 72hr

PDF file - 40K, Time activity curves for 111In-labeled 2G10 and 3C6 in MDA-MB-231 orthotopic xenograft tumor-bearing mice

PDF file - 58K, 177Lu-2G10 SPECT/CT imaging of a mouse enrolled in the RIT study. The mouse was injected with 2.5�g of 2G10 labeled with 75°Ci of 177Lu. The image shown is a coronal view acquitted at 24hrs post-injection. The represented data was processed using AMIDE software

Introduction In this study, a structurally modified phosphoramidate scaffold, with improved prostate-specific membrane antigen (PSMA) avidity, stability and in vivo characteristics, as a PET imaging agent for prostate cancer (PCa), was prepared and evaluated. Methods p-Fluorobenzoyl-aminohexanoate and 2-(3-hydroxypropyl)glycine were introduced into the PSMA-targeting scaffold yielding phosphoramidate 5. X-ray crystallography was performed on the PSMA/5 complex. [ 18 F]5 was synthesized, and cell uptake and internalization studies were conducted in PSMA(+) LNCaP and CWR22Rv1 cells and PSMA(−) PC-3 cells. In vivo PET imaging and biodistribution studies were performed at 1 and 4h post injection in mice bearing CWR22Rv1 tumor, with or without blocking agent. Results The crystallographic data showed interaction of the p -fluorobenzoyl group with an arene-binding cleft on the PSMA surface. In vitro studies revealed elevated uptake of [ 18 F]5 in PSMA(+) cells (2.2% in CWR22Rv1 and 12.1% in LNCaP) compared to PSMA(−) cells (0.08%) at 4h. In vivo tumor uptake of 2.33% ID/g and tumor-to-blood ratio of 265:1 was observed at 4h. Conclusions We have successfully synthesized, radiolabeled and evaluated a new PSMA-targeted PET agent. The crystal structure of the PSMA/5 complex highlighted the interactions within the arene-binding cleft contributing to the overall complex stability. The high target uptake and rapid non-target clearance exhibited by [ 18 F]5 in PSMA(+) xenografts substantiates its potential use for PET imaging of PCa. Advances in Knowledge The only FDA-approved imaging agent for PCa, Prostascint®, targets PSMA but suffers from inherent shortcomings. The data acquired in this manuscript confirmed that our new generation of [ 18 F]-labeled PSMA inhibitor exhibited promising in vivo performance as a PET imaging agent for PCa and is well-positioned for subsequent clinical trials. Implications for Patient Care Our preliminary data demonstrate that this tracer possesses the required imaging characteristics to be sensitive and specific for PCa imaging in patients at all stages of the disease.

Subtype-targeted therapies can have a dramatic impact on improving the quality and quantity of life for women suffering from breast cancer. Despite an initial therapeutic response, cancer recurrence and acquired drug-resistance are commonplace. Non-invasive imaging probes that identify drug-resistant lesions are urgently needed to aid in the development of novel drugs and the effective utilization of established therapies for breast cancer. The protease receptor urokinase plasminogen activator receptor (uPAR) is a target that can be exploited for non-invasive imaging. The expression of uPAR has been associated with phenotypically aggressive breast cancer and acquired drug-resistance. Acquired drug-resistance was modeled in cell lines from two different breast cancer subtypes, the uPAR negative luminal A subtype and the uPAR positive triple negative subtype cell line MDA-MB-231. MCF-7 cells, cultured to be resistant to tamoxifen (MCF-7 TamR), were found to significantly over-express uPAR compared to the parental cell line. uPAR expression was maintained when resistance was modeled in triple-negative breast cancer by generating doxorubicin and paclitaxel resistant MDA-MB-231 cells (MDA-MB-231 DoxR and MDA-MB-231 TaxR). Using the antagonistic uPAR antibody 2G10, uPAR was imaged in vivo by near-infrared (NIR) optical imaging and (111)In-single photon emission computed tomography (SPECT). Tumor uptake of the (111)In-SPECT probe was high in the three drug-resistant xenografts (46 %ID/g) and minimal in uPAR negative xenografts at 72 hours post-injection. This preclinical study demonstrates that uPAR can be targeted for imaging breast cancer models of acquired resistance leading to potential clinical applications.

Components of the plasminogen activation system, which are overexpressed in aggressive breast cancer subtypes, offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared imaging and 111In single-photon emission computed tomography. Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard 18F-fluorodeoxyglucose at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. In addition, a radioimmunotherapy study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope 177Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof of concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology. Cancer Res; 73(7); 2070–81. ©2013 AACR.

The fields of nanotechnology and medicine have merged in the development of new imaging and drug delivery agents based on nanoparticle platforms. As one example, a mutant of bacteriophage MS2 can be differentially modified on the exterior and interior surfaces for the concurrent display of targeting functionalities and payloads, respectively. In order to realize their potential for use in in vivo applications, the biodistribution and circulation properties of this class of agents must first be investigated. A means of modulating and potentially improving the characteristics of nanoparticle agents is the appendage of PEG chains. Both MS2 and MS2-PEG capsids possessing interior DOTA chelators were labeled with (64)Cu and injected intravenously into mice possessing tumor xenografts. Dynamic imaging of the agents was performed using PET-CT on a single animal per sample, and the biodistribution at the terminal time point (24 h) was assessed by gamma counting of the organs ex vivo for 3 animals per agent. Compared to other viral capsids of similar size, the MS2 agents showed longer circulation times. Both MS2 and MS2-PEG bacteriophage behaved similarly, although the latter agent showed significantly less uptake in the spleen. This effect may be attributed to the ability of the PEG chains to mask the capsid charge. Although the tumor uptake of the agents may result from the enhanced permeation and retention (EPR) effect, selective tumor imaging may be achieved in the future by using exterior targeting groups.

A variety of nanoscale scaffolds, including virus-like particles (VLPs), are being developed for biomedical applications; however, little information is available about their in vivo behavior. Targeted nanoparticles are particularly valuable as diagnostic and therapeutic carriers because they can increase the signal-to-background ratio of imaging agents, improve the efficacy of drugs, and reduce adverse effects by concentrating the therapeutic molecule in the region of interest. The genome-free capsid of bacteriophage MS2 has several features that make it well-suited for use in delivery applications, such as facile production and modification, the ability to display multiple copies of targeting ligands, and the capacity to deliver large payloads. Anti-EGFR antibodies were conjugated to MS2 capsids to construct nanoparticles targeted toward receptors overexpressed on breast cancer cells. The MS2 agents showed good stability in physiological conditions up to 2 days and specific binding to the targeted receptors in in vitro experiments. Capsids radiolabeled with

SEE DREIER DOI 101093/AWW112 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: For many decades a breakdown of the blood-brain barrier has been postulated to occur in migraine. Hypothetically this would facilitate access of medications, such as dihydroergotamine or triptans, to the brain despite physical properties otherwise restricting their entry. We studied the permeability of the blood-brain barrier in six migraineurs and six control subjects at rest and during acute glyceryl trinitrate-induced migraine attacks using positron emission tomography with the novel radioligand (11)C-dihydroergotamine, which is chemically identical to pharmacologically active dihydroergotamine. The influx rate constant Ki, average dynamic image and time activity curve were assessed using arterial blood sampling and served as measures for receptor binding and thus blood-brain barrier penetration. At rest, there was binding of (11)C-dihydroergotamine in the choroid plexus, pituitary gland, and venous sinuses as expected from the pharmacology of dihydroergotamine. However, there was no binding to the brain parenchyma, including the hippocampus, the area with the highest density of the highest-affinity dihydroergotamine receptors, and the raphe nuclei, a postulated brainstem site of action during migraine, suggesting that dihydroergotamine is not able to cross the blood-brain barrier. This binding pattern was identical in migraineurs during glyceryl trinitrate-induced migraine attacks as well as in matched control subjects. We conclude that (11)C-dihydroergotamine is unable to cross the blood-brain barrier interictally or ictally demonstrating that the blood-brain barrier remains tight for dihydroergotamine during acute glyceryl trinitrate-induced migraine attacks.

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with 111In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor–mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer–associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions. Cancer Res; 75(7); 1225–35. ©2015 AACR.

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in ≈30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-β receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans , which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.

Using longitudinal micro positron emission tomography (microPET)/computed tomography (CT) studies, we quantified changes in myocardial metabolism and perfusion in spontaneously hypertensive rats (SHRs), a model of left ventricular hypertrophy (LVH). Fatty acid and glucose metabolism were quantified in the hearts of SHRs and Wistar-Kyoto (WKY) normotensive rats using long-chain fatty acid analog 18 F-fluoro-6-thia heptadecanoic acid ( 18 F-FTHA) and glucose analog 18 F-fluorodeoxyglucose ( 18 F-FDG) under normal or fasting conditions. We also used 18 F-fluorodihydrorotenol ( 18 F-FDHROL) to investigate perfusion in their hearts without fasting. Rats were imaged at 4 or 5 times over their life cycle. Compartment modeling was used to estimate the rate constants for the radiotracers. Blood samples were obtained and analyzed for glucose and free fatty acid concentrations. SHRs demonstrated no significant difference in 18 F-FDHROL wash-in rate constant ( P = .1) and distribution volume ( P = .1), significantly higher 18 F-FDG myocardial influx rate constant ( P = 4×10 −8 ), and significantly lower 18 F-FTHA myocardial influx rate constant ( P = .007) than WKYs during the 2009-2010 study without fasting. SHRs demonstrated a significantly higher 18 F-FDHROL wash-in rate constant ( P = 5×10 −6 ) and distribution volume ( P = 3×10 −8 ), significantly higher 18 F-FDG myocardial influx rate constant ( P = 3×10 −8 ), and a higher trend of 18 F-FTHA myocardial influx rate constant (not significant, P = .1) than WKYs during the 2011–2012 study with fasting. Changes in glucose plasma concentrations were generally negatively correlated with corresponding radiotracer influx rate constant changes. The study indicates a switch from preferred fatty acid metabolism to increased glucose metabolism with hypertrophy. Increased perfusion during the 2011-2012 study may be indicative of increased aerobic metabolism in the SHR model of LVH.

Due to unfavorable molecular size and lipophilicity, migraine-specific medications such as dihydroergotamine (DHE) are not expected to penetrate the blood-brain barrier (BBB). A breakdown of the BBB during migraine attacks has been postulated as the mechanism in which DHE accesses postulated central sites of action.

Radiosynthesis of a fluorine-18 labeled organophosphate (OP) inhibitor of acetylcholinesterase (AChE) and subsequent positron emission tomography (PET) imaging using the tracer in the rat central nervous system are reported. The tracer structure, which contains a novel β-fluoroethoxy phosphoester moiety, was designed as an insecticide-chemical nerve agent hybrid to optimize handling and the desired target reactivity. Radiosynthesis of the β-fluoroethoxy tracer is described that utilizes a [(18)F]prosthetic group coupling approach. The imaging utility of the [(18)F]tracer is demonstrated in vivo within rats by the evaluation of its brain penetration and cerebral distribution qualities in the absence and presence of a challenge agent. The tracer effectively penetrates brain and localizes to cerebral regions known to correlate with the expression of the AChE target. Brain pharmacokinetic properties of the tracer are consistent with the formation of an OP-adducted acetylcholinesterase containing the fluoroethoxy tracer group. Based on the initial favorable in vivo qualities found in rat, additional [(18)F]tracer studies are ongoing to exploit the technology to dynamically probe organophosphate mechanisms of action in mammalian live tissues.

UnlabelledMyocardial metabolic and perfusion imaging is a vital tool for understanding the physiologic consequences of heart failure. We used PET imaging to examine the longitudinal kinetics of (18)F-FDG and 14(R,S)-(18)F-fluoro-6-thia-heptadecanoic acid ((18)F-FTHA) as analogs of glucose and fatty acid (FA) to quantify metabolic substrate shifts with the spontaneously hypertensive rat (SHR) as a model of left ventricular hypertrophy (LVH) and failure. Myocardial perfusion and left ventricular function were also investigated using a newly developed radiotracer (18)F-fluorodihydrorotenol ((18)F-FDHROL).MethodsLongitudinal dynamic electrocardiogram-gated small-animal PET/CT studies were performed with 8 SHR and 8 normotensive Wistar-Kyoto (WKY) rats over their life cycle. We determined the myocardial influx rate constant for (18)F-FDG and (18)F-FTHA (Ki(FDG) and Ki(FTHA), respectively) and the wash-in rate constant for (18)F-FDHROL (K1(FDHROL)). (18)F-FDHROL data were also used to quantify left ventricular ejection fraction (LVEF) and end-diastolic volume (EDV). Blood samples were drawn to independently measure plasma concentrations of glucose, insulin, and free fatty acids (FFAs).ResultsKi(FDG) and Ki(FTHA) were higher in SHRs than WKY rats (P < 3 × 10(-8) and 0.005, respectively) independent of age. A decrease in Ki(FDG) with age was evident when models were combined (P = 0.034). The SHR exhibited higher K1(FDHROL) (P < 5 × 10(-6)) than the control, with no age-dependent trends in either model (P = 0.058). Glucose plasma concentrations were lower in SHRs than controls (P < 6 × 10(-12)), with an age-dependent rise for WKY rats (P < 2 × 10(-5)). Insulin plasma concentrations were higher in SHRs than controls (P < 3 × 10(-3)), with an age-dependent decrease when models were combined (P = 0.046). FFA levels were similar between models (P = 0.374), but an increase with age was evident only in SHR (P < 7 × 10(-6)).ConclusionThe SHR exhibited alterations in myocardial substrate use at 8 mo characterized by increased glucose and FA utilizations. At 20 mo, the SHR had LVH characterized by decreased LVEF and increased EDV, while simultaneously sustaining higher glucose and similar FA utilizations (compared with WKY rats), which indicates maladaptation of energy substrates in the failing heart. Elevated K1(FDHROL) in the SHR may reflect elevated oxygen consumption and decreased capillary density in the hypertrophied heart. From our findings, metabolic changes appear to precede mechanical changes of LVH progression in the SHR model.

Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site–specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, 111 In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1–dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.

[(124)I]m-iodobenzylguanidine ((124)I-mIBG) provides a quantitative tool for pretherapy tumor imaging and dosimetry when performed before [(131)I]m-iodobenzylguanidine ((131)I-mIBG) targeted radionuclide therapy of neuroblastoma. (124)I (T (1/2) = 4.2 days) has a comparable half-life to that of (131)I (T (1/2) = 8.02 days) and can be imaged by positron emission tomography (PET) for accurate quantification of the radiotracer distribution. We estimated expected radiation dose in tumors from (131)I-mIBG therapy using (124)I-mIBG microPET/CT imaging data in a murine xenograft model of neuroblastoma transduced to express high levels of the human norepinephrine transporter (hNET).In order to enhance mIBG uptake for in vivo imaging and therapy, NB 1691-luciferase (NB1691) human neuroblastoma cells were engineered to express high levels of hNET protein by lentiviral transduction (NB1691-hNET). Both NB1691 and NB1691-hNET cells were implanted subcutaneously and into renal capsules in athymic mice. (124)I-mIBG (4.2-6.5 MBq) was administered intravenously for microPET/CT imaging at 5 time points over 95 h (0.5, 3-5, 24, 48, and 93-95 h median time points). In vivo biodistribution data in normal organs, tumors, and whole-body were collected from reconstructed PET images corrected for photon attenuation using the CT-based attenuation map. Organ and tumor dosimetry were determined for (124)I-mIBG. Dose estimates for (131)I-mIBG were made, assuming the same in vivo biodistribution as (124)I-mIBG.All NB1691-hNET tumors had significant uptake and retention of (124)I-mIBG, whereas unmodified NB1691 tumors did not demonstrate quantifiable mIBG uptake in vivo, despite in vitro uptake. (124)I-mIBG with microPET/CT provided an accurate three-dimensional tool for estimating the radiation dose that would be delivered with (131)I-mIBG therapy. For example, in our model system, we estimated that the administration of (131)I-mIBG in the range of 52.8-206 MBq would deliver 20 Gy to tumors.The overexpression of hNET was found to be critical for (124)I-mIBG uptake and retention in vivo. The quantitative (124)I-mIBG PET/CT is a promising new tool to predict tumor radiation doses with (131)I-mIBG therapy of neuroblastoma. This methodology may be applied to tumor dosimetry of (131)I-mIBG therapy in human subjects using (124)I-mIBG pretherapy PET/CT data.