Thomas D. Harris

Catalyzed reporter deposition, a novel method of signal amplification: application to immunoassays

A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using a horseradish peroxidase (HRP) ADRE to catalyze the deposition of biotin labeled phenols. The deposited biotins are then reacted with streptavidin-labeled enzyme, thereby resulting in deposition of enzyme. Using the ADRE to catalyze the deposition of additional enzyme results in an amplification of the signal of the ADRE alone and improves the detection limit of the assay. The method is highly sensitive, simple, flexible, and easy to implement.

Endothelial ανβ3 Integrin–Targeted Fumagillin Nanoparticles Inhibit Angiogenesis in Atherosclerosis

Objective—Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. &agr;&ngr;&bgr;3 Integrin–targeted paramagnetic nanoparticles were prepared for noninvasive assessment of angiogenesis in early atherosclerosis, for site-specific delivery of antiangiogenic drug, and for quantitative follow-up of response. Methods and Results—Expression of &agr;&ngr;&bgr;3 integrin by vasa vasorum was imaged at 1.5 T in cholesterol-fed rabbit aortas using integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 &mgr;g/kg or 30 &mgr;g/kg. Both formulations produced similar MRI signal enhancement (16.7%±1.1%) when integrated across all aortic slices from the renal arteries to the diaphragm. Seven days after this single treatment, integrin-targeted paramagnetic nanoparticles were readministered and showed decreased MRI enhancement among fumagillin-treated rabbits (2.9%±1.6%) but not in untreated rabbits (18.1%±2.1%). In a third group of rabbits, nontargeted fumagillin nanoparticles did not alter vascular &agr;&ngr;&bgr;3-integrin expression (12.4%±0.9%; P>0.05) versus the no-drug control. In a second study focused on microscopic changes, fewer microvessels in the fumagillin-treated rabbit aorta were counted compared with control rabbits. Conclusions—This study illustrates the potential of combined molecular imaging and drug delivery with targeted nanoparticles to noninvasively define atherosclerotic burden, to deliver effective targeted drug at a fraction of previous levels, and to quantify local response to treatment.

Molecular MR imaging of melanoma angiogenesis with ανβ3-targeted paramagnetic nanoparticles

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether ανβ3‐targeted paramagnetic nanoparticles can detect and characterize sparse ανβ integrin expression on neovasculature induced by nascent melanoma xenografts (∼30 mm3) at 1.5T. Athymic nude mice bearing human melanoma tumors were intravenously injected with αvβ3‐integrin‐targeted paramagnetic nanoparticles, nontargeted paramagnetic nanoparticles, or αvβ3‐targeted‐nonparamagnetic nanoparticles 2 hr before they were injected with αvβ3‐integrin‐targeted paramagnetic nanoparticles (i.e., in vivo competitive blockade) and imaged with MRI. Contrast enhancement of neovascularity in animals that received ανβ3‐targeted paramagnetic nanoparticles increased 173% by 120 min. Signal contrast with nontargeted paramagnetic nanoparticles was approximately 50% less than that in the targeted group (P < 0.05). Molecular MRI results were corroborated by histology. In a competitive cell adhesion assay, incubation of ανβ3‐expressing cells with targeted nanoparticles significantly inhibited binding to a vitronectin‐coated surface, confirming the bioactivity of the targeted nanoparticles. The present study lowers the limit previously reported for detecting sparse biomarkers with molecular MRI in vivo. This technique may be employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumors, and to phenotype and stage early melanoma in a clinical setting. Magn Reson Med 53:621–627, 2005.

Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction.

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.

Detection of Injury-Induced Vascular Remodeling by Targeting Activated αvβ3 Integrin In Vivo

Background—The &agr;vβ3 integrin plays a critical role in cell proliferation and migration. We hypothesized that vascular cell proliferation, a hallmark of injury-induced remodeling, can be tracked by targeting &agr;vβ3 integrin expression in vivo. Methods and Results—RP748, a novel 111In-labeled &agr;vβ3-specific radiotracer, was evaluated for its cell-binding characteristics and ability to track injury-induced vascular proliferation in vivo. Three groups of experiments were performed. In cultured endothelial cells (ECs), TA145, a cy3-labeled homologue of RP748, localized to &agr;vβ3 at focal contacts. Activation of&agr;vβ3 by Mn 2+ led to increased EC binding of TA145. Left common carotid artery wire injury in apolipoprotein E−/− mice led to vascular wall expansion over a period of 4 weeks. RP748 (7.4 MBq) was injected into groups of 9 mice at 1, 3, or 4 weeks after left carotid injury, and carotids were harvested for autoradiography. Relative autographic intensity, defined as counts/pixel of the injured left carotid area divided by counts/pixel of the uninjured right carotid area, was higher at 1 and 3 weeks (1.8±0.1 and 1.9±0.2, respectively) and decreased significantly by 4 weeks after injury (1.4±0.1, P <0.05). Carotid &agr;v and β3 integrin expression was maximal at 1 week and decreased by 4 weeks after injury. The proliferation index, as determined by Ki67 staining, followed a temporal pattern similar to that of RP748 uptake. Dynamic gamma imaging demonstrated rapid renal clearance of RP748. Conclusions—RP748 has preferential binding to activated &agr;vβ3 integrin and can track the injury-induced vascular proliferative process in vivo.

Design, Synthesis, and Evaluation of Radiolabeled Integrin αvβ3 Receptor Antagonists for Tumor Imaging and Radiotherapy

The goal of this research is the development of tumor imaging and radiotherapeutic agents based on targeting of the integrin αvβ3 (vitronectin receptor). Macrocyclic chelator DOTA has been conjugated to peptidomimetic vitronectin receptor antagonist SH066 to give TA138. TA138 and 89Y-TA138 retain antagonist properties and high affinity for integrin αvβ3 (IC50 = 12 and 18 nM, respectively), and good selectivity versus integrin αIIbβ3 (IC50 > 10,000 nM). TA138 forms stable complexes with 111In and 90Y in > 95% RCP. 111In-TA138 demonstrates high tumor uptake in the c-neu Oncomouse® (Charles River Laboratories [Charles River, Canada]) mammary adenocarcinoma model (9.39% ID/g at 2 hours PI) and low background activity. Blood clearance is rapid and excretion is renal. Tumors are visible as early as 0.5 hours PI. Radiotherapy studies in the c-neu Oncomouse® model demonstrated a slowing of tumor growth at a dose of 15 mCi/m2, and a regression of tumors at a dose of 90 mCi/m2.

MR three-dimensional molecular imaging of intramural biomarkers with targeted nanoparticles.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Radiolabeled divalent peptidomimetic vitronectin receptor antagonists as potential tumor radiotherapeutic and imaging agents.

The integrin receptor alphavbeta3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled alphavbeta3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin alphavbeta3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 A) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.

αvβ3-Targeted detection of arteriopathy in transplanted human coronary arteries: an autoradiographic study

Graft arteriopathy (GA), characterized by diffuse concentric narrowing of coronary arteries, is the major cause of late graft failure in cardiac transplantation. αvβ3 Integrin is up‐regulated in proliferating vascular cells and may constitute an appropriate target for imaging GA. We used a human/mouse chimeric model of GA, in which segments of human coronary artery were transplanted to severe combined immunodeficiency mice, followed by reconstitution with allogeneic human peripheral blood mononuclear cells (PBMC). This led to vascular remodeling characterized by neointima formation over a period of 4 wk. αvβ3 expression in the graft was minimal in animals without PBMC, considerably increased by 2 wk, and decreased toward baseline by 4 wk after PBMC reconstitution. Cell proliferation was maximal at 2 wk, correlating with peak αvβ3 expression. RP748, an 111In‐labeled αvβ3 (active conformation)‐targeted radiotracer was injected into groups of 5 recipients at 0, 2, and 4 wk after PBMC reconstitution. Relative uptakes, defined as autoradiographic intensity in the graft/native aortas closely tracked the proliferative process. Specificity of uptake was demonstrated using excess nonlabeled tracer. In conclusion, αvβ3 integrin is transiently up‐regulated (and activated) in GA and may be targeted by RP748 for detection of the proliferative process in early GA.

Synthesis, evaluation and Tc-99m complexation of a hydrazinonicotinyl conjugate of a GP IIb/IIIa antagonist cyclic peptide for the detection of deep vein thrombosis

Abstract A cyclic peptide GP IIb/IIIa receptor antagonist containing the N-Me-Arg-Gly-Asp motif has been derivatized with the technetium chelating hydrazinonicotinyl group (Hynic). The Hynic derivative, and the Tc-99 diazenido complex, retain the high receptor affinity of the parent peptide. The Tc-99m complex shows high thrombus uptake, and rapid clearance of background, producing excellent images in under 1 h.