Marina V. Backer

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

Vascular endothelial growth factor selectively targets boronated dendrimers to tumor vasculature

Tumor neovasculature is a potential but, until very recently, unexplored target for boron neutron capture therapy (BNCT) of cancer. In the present report, we describe the construction of a vascular endothelial growth factor (VEGF)–containing bioconjugate that potentially could be used to target up-regulated VEGF receptors (VEGFR), which are overexpressed on tumor neovasculature. A fifth-generation polyamidoamine dendrimer containing 128 reactive amino groups was reacted with 105 to 110 decaborate molecules to produce a macromolecule with 1,050 to 1,100 boron atoms per dendrimer. This was conjugated to thiol groups of VEGF at a 4:1 molar ratio using the heterobifunctional reagent sulfo-LC-SPDP. In addition, the boronated dendrimer was tagged with a near-IR Cy5 dye to allow for near-IR fluorescent imaging of the bioconjugate in vitro and in vivo. As would be predicted, the resulting VEGF-BD/Cy5 bioconjugate was not cytotoxic to HEK293 cells engineered to express 2.5 × 106 VEGFR-2 per cell. Furthermore, it showed binding and activation of VEGFR-2 comparable with that of native VEGF. Internalization of VEGF-BD/Cy5 by PAE cells expressing 2.5 × 105 VEGFR-2 per cell was inhibited by excess VEGF, indicating a VEGFR-2-mediated mechanism of uptake. Near-IR fluorescent imaging of 4T1 mouse breast carcinoma revealed selective accumulation of VEGF-BD/Cy5, but not BD/Cy5, particularly at the tumor periphery where angiogenesis was most active. Accumulation of VEGF-BD/Cy5 in 4T1 breast carcinoma was diminished in mice pretreated with a toxin-VEGF fusion protein that selectively killed VEGFR-2-overexpressing endothelial cells. Our data lay the groundwork for future studies using the VEGF-BD/Cy5 bioconjugate as a targeting agent for BNCT of tumor neovasculature.

scVEGF Microbubble Ultrasound Contrast Agents: A Novel Probe for Ultrasound Molecular Imaging of Tumor Angiogenesis

Objective:To develop a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single-chain vascular endothelial growth factor construct (scVEGF) through uniform site-specific conjugation for ultrasound imaging of tumor angiogenesis. Methods:Ligand conjugation to maleimide-bearing MB by thioether bonding was first validated with a fluorophore (BODIPY-cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MBs were subsequently site-specifically conjugated to cysteine-containing Cys-tag in scVEGF, and bound scVEGF was quantified by enzyme-linked immunosorbent assay. Targeted adhesion of scVEGF-MB was investigated with in vitro parallel plate flow chamber assays with recombinant murine VEGFR-2 substrates and human VEGFR-2-expressing porcine endothelial cells (PAE/KDR). A wall-less ultrasound flow phantom, with flow channels coated with immobilized VEGFR-2, was used to detect adhesion of scVEGF-MB with contrast ultrasound imaging. A murine model of colon adenocarcinoma was used to assess retention of scVEGF-MB with contrast ultrasound imaging during tumor angiogenesis in vivo. Results:Proof-of-principle of ligand conjugation to maleimide-bearing MB was demonstrated with a BODIPY-cysteine fluorophore. Conjugation of BODIPY to MB saturated at 10-fold molar excess BODIPY relative to maleimide groups on MB surfaces. MB reacted with scVEGF and led to the conjugation of 1.2 × 105 molecules scVEGF per MB. Functional adhesion of sc-VEGF-MB was shown in parallel plate flow chamber assays. At a shear stress of 1.0 dynes/cm2, scVEGF-MB exhibited 5-fold higher adhesion to both recombinant VEGFR-2 substrates and VEGFR-2-expressing endothelial cells compared with nontargeted control MB. Additionally, scVEGF-MB targeted to immobilized VEGFR-2 in an ultrasound flow phantom showed an 8-fold increase in mean acoustic signal relative to casein-coated control channels. In an in vivo model of tumor angiogenesis, scVEGF MB showed significantly higher ultrasound contrast signal enhancement in tumors (8.46 ± 1.61 dB) compared with nontargeted control MB (1.58 ± 0.83 dB). Conclusions:These results demonstrate the functionality of a novel scVEGF-bearing MB contrast agent, which could be useful for molecular imaging of VEGFR-2 in basic science and drug discovery research.

Boron Containing Macromolecules and Nanovehicles as Delivery Agents for Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when non-radioactive boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer (LET) alpha particles ((4)He) and recoiling lithium -7((7)Li) nuclei. For BNCT to be successful, a sufficient number of (10)B atoms ( approximately 10(9) atoms/cell) must be selectively delivered to the tumor and enough thermal neutrons must be absorbed by them to sustain a lethal (10)B(n, alpha) (7)Li capture reaction. BNCT primarily has been used to treat patients with brain tumors, and more recently those with head and neck cancer. Two low molecular weight (LMW) boron delivery agents currently are being used clinically, sodium borocaptate and boronophenylalanine. However, a variety of high molecular weight (HMW) agents consisting of macromolecules and nanovehicles have been developed. This review will focus on the latter which include: monoclonal antibodies, dendrimers, liposomes, dextrans, polylysine, avidin, folic acid, and epidermal and vascular endothelial growth factors (EGF and VEGF). Procedures for introducing boron atoms into these HMW agents and their chemical properties will be discussed. In vivo studies on their biodistribution will be described, and the efficacy of a subset of them, which have been used for BNCT of tumors in experimental animals, will be discussed. Since brain tumors currently are the primary candidates for treatment by BNCT, delivery of these HMW agents across the blood-brain barrier presents a special challenge. Various routes of administration will be discussed including receptor-facilitated transcytosis following intravenous administration, direct intratumoral injection and convection enhanced delivery by which a pump is used to apply a pressure gradient to establish bulk flow of the HMW agent during interstitial infusion. Finally, we will conclude with a discussion relating to issues that must be addressed if these HMW agents are to be used clinically.

Analysis of In Situ and Ex Vivo Vascular Endothelial Growth Factor Receptor Expression During Experimental Aortic Aneurysm Progression

Objective—Mural inflammation and neovascularization are characteristic pathological features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E–deficient (Apo E−/−) murine AAA model. Methods and Results—Apo E−/− mice maintained on a high-fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n=16 mice, 3 to 4 time points per mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n=9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy5.5 fluorescent tracer (7 to 18 &mgr;g/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 &mgr;g/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression. Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31+ cell density. Conclusion—Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.

ScVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA conjugates: comparison of easy-to-label recombinant proteins for [68Ga]PET imaging of VEGF receptors in angiogenic vasculature

INTRODUCTION VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with (68)Ga at ambient temperature. METHODS scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)](-) complex with N-hydroxysuccinimide. Reaction of the activated complex with NH(2)-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH(2)-Bn-NOTA. (68)Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection. RESULTS The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of (68)Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h. CONCLUSIONS The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy-to-perform labeling with high stability for fast [(68)Ga]PET imaging of VEGF receptors in angiogenic vasculature.

Imaging key biomarkers of tumor angiogenesis.

Angiogenesis is a fundamental requirement for tumor growth and therefore it is a primary target for anti-cancer therapy. Molecular imaging of angiogenesis may provide novel opportunities for early diagnostic and for image-guided optimization and management of therapeutic regimens. Here we reviewed the advances in targeted imaging of key biomarkers of tumor angiogenesis, integrins and receptors for vascular endothelial growth factor (VEGF). Tracers for targeted imaging of these biomarkers in different imaging modalities are now reasonably well-developed and PET tracers for integrin imaging are currently in clinical trials. Molecular imaging of longitudinal responses to anti-angiogenic therapy in model tumor systems revealed a complex pattern of changes in targeted tracer accumulation in tumor, which reflects drug-induced tumor regression followed by vascular rebound. Further work will define the competitiveness of targeted imaging of key angiogenesis markers for early diagnostic and image-guided therapy.

In Vivo, Dual-Modality OCT/LIF Imaging Using a Novel VEGF Receptor-Targeted NIR Fluorescent Probe in the AOM-Treated Mouse Model

PurposeIncreased vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis, particularly in tumor growth areas, as compared with quiescent vasculature. An increase in VEGF receptor-2 is associated with colon cancer progression. The in vivo detection of VEGF receptor is of interest for the purposes of studying basic mechanisms of carcinogenesis, making clinical diagnoses, and monitoring the efficacy of chemopreventive and therapeutic agents. In this study, a novel single chain (sc)VEGF-based molecular probe is utilized in the azoxymethane (AOM)-treated mouse model of colorectal cancer to study delivery route and specificity for disease.ProceduresThe probe was constructed by site-specific conjugation of a near-infrared fluorescent dye, Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography/laser-induced fluorescence (OCT/LIF) endoscopic system. A probe inactivated via excessive biotinylation was utilized as a control for nonreceptor-mediated binding. The LIF excitation source was a 633-nm He:Ne laser, and red/near-infrared fluorescence was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo. OCT-detected disease was verified with hematoxylin and eosin stained histology slides ex vivo.ResultsHigh fluorescence emission intensity from the targeted probe was correlated with tumor presence as detected using OCT in vivo and VEGFR-2 immunostaining on histological sections ex vivo. The inactivated probe accumulated preferentially on the surface of tumor lesions and in lymphoid aggregate tissue and was less selective for VEGFR-2.ConclusionThe scVEGF/Cy probe delivered via colonic lavage reaches tumor vasculature and selectively accumulates in VEGFR-2-positive areas, resulting in high sensitivity and specificity for tumor detection. The combination of OCT and LIF imaging modalities may allow the simultaneous study of tumor morphology and protein expression for the development of diagnostic and therapeutic methods for colorectal cancer.

Direct site-specific labeling of the Cys-tag moiety in scVEGF with technetium 99m.

Angiogenesis is a fundamental feature of tumor development, and therefore, the tracers for molecular imaging of specific angiogenic biomarkers are expected to be useful for diagnostics, patient monitoring, and drug development. We have created a new class of imaging agents based on the most important mediator of angiogenesis, vascular endothelial growth factor (VEGF). Our latest version is a single-chain (sc) VEGF protein containing an N-terminal Cys-tag designed for site-specific modification with a variety of imaging and therapeutic moieties. We have recently found that the Cys-tag itself can form a stable chelate with (99m)Tc using tin-tricine as an exchange reagent. This self-chelation approach yields a highly stable and fully functional form of radiolabeled scVEGF that can be used as a SPECT tracer for tumor angiogenesis. Also of note is that directly labeled scVEGF has less than one-half the nonspecific renal uptake of (99m)Tc-HYNIC-scVEGF. The simple production of scVEGF for direct chelation of (99m)Tc makes it a promising molecular imaging agent for the oncology clinic.

Targeting the unfolded protein response in cancer therapy.

Rapid growth of tumor cells coupled with inadequate vascularization leads to shortage of oxygen and nutrients. The unfolded protein response (UPR), a defense cellular mechanism activated during such stress conditions, is a complex process that includes upregulation of the endoplasmic reticulum chaperones, such as glucose-regulated protein 78 (GRP78). Due to its central role in UPR, GRP78 is overexpressed in many cancers; it is implicated in cancer cell survival through supporting of drug- and radioresistance as well as metastatic dissemination, and is generally associated with poor outcome. This is the reason why selective destruction of GRP78 could become a novel anticancer strategy. GRP78 is the only known substrate of the proteolytic A subunit (SubA) of a bacterial AB(5) toxin, and the selective SubA-induced cleavage of GRP78 leads to massive cell death. Targeted delivery of SubA into cancer cells via specific receptor-mediated endocytosis could be a suitable strategy for assaulting tumor cells. We fused SubA to epidermal growth factor (EGF), whose receptor (EGFR) is frequently overexpressed in tumor cells, and demonstrated that the resulting EGF-SubA immunotoxin is an effective killer of EGFR-positive tumor cells. Furthermore, because of its unique mechanism of action, EGF-SubA synergizes with UPR-inducing drugs, which opens a possibility for the development of mechanism-based combination regimens for effective anticancer therapy. In this chapter, we provide experimental protocols for the assessment of the effects of EGF-SubA on EGFR-positive cancer cells, either alone or in combination with UPR-inducing drugs.