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Diagnosis of glioblastoma multiform (GBM) with MRI lacks molecular information and requires a biopsy for pathologic confirmation. The EGFRvIII, is a constitutively active mutant of the EGF receptor, identified in a high percentage of brain cancers and associated with increased invasiveness and resistance, making it a good target to improve imaging and diagnosis. The present study shows that conjugation of near-infrared quantum dot (Qd800) to an anti-EGFRvIII single domain antibody, made of the variable region with an extra cysteine for site-specific conjugation (EG2-Cys), increased its internalization in U87MG-EGFRvIII cells in vitro compared to Qd800 conjugated with the Fc region of the antibody (EG2-hFc) or unconjugated. EG2-Cys also improved the contrast in Near-Infrared Imaging of mice bearing orthotopic glioblastoma. The increased accumulation was confirmed by fluorescence microscopy of brain sections. The specificity of EG2-Cys in brain tumor expressing the EGFRvIII mutant receptor may provide an accurate less invasive diagnosis and determine the level of tumor aggressiveness and resistance.

Background: Glioma is the most common and most difficult to treat brain cancer. Despite many efforts treatment, efficacy remains low. As neurosurgical removal is the standard procedure for glioma, a method, allowing for both early detection and exact determination of the location, size and extent of the tumor, could improve a patient's positive response to therapy., New Method: We propose application of susceptibility weighted molecular magnetic resonance imaging using, targeted contrast agents, based on superparamagnetic iron oxide nanoparticles, for imaging of the, glioma rim, namely brain-tumor interface. Iron oxide attached to the targeted cells increases, susceptibility differences at the boundary between tumor and normal tissue, providing the opportunity, to utilize susceptibility weighted imaging for improved tumor delineation. We investigated potential, enhancement of the tumor-brain contrast, including tumor core and rim when using susceptibility, weighted MRI for molecular imaging of glioma., Results: There were significant differences in contrast-to-noise ratio before, 12 and 120min after contrast, agent injection between standard gradient echo pulse sequence and susceptibility weighted molecular, magnetic resonance imaging for the core-brain, tumor rim-core and tumor rim-brain areas., Comparison With Existing Methods: Currently, the most common MRI contrast agent used for glioma diagnosis is a non-specific, gadolinium-based agent providing T1-weighted enhancement. Susceptibility-weighted magnetic, resonance imaging is much less efficient when no targeted superparamagnetic contrast agents are, used., Conclusion: The improved determination of glioma extent provided by SWI offers an important new tool for, diagnosis and surgical planning., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1μg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A mouse model of glioblastoma multiforme was used to determine the accumulation of a targeted contrast agent in tumor vessels. The contrast agent, consisting of superparamagnetic iron oxide coated with dextran, was functionalized with an anti-insulin-like-growth-factor binding protein 7 (anti-IGFBP7) single domain antibody. The near infrared marker, Cy5.5, was also attached for an in vivo fluorescence study. A 9.4T magnetic resonance imaging (MRI) system was used for in vivo studies on days 10 and 11 following tumor inoculation. T(2) relaxation time was used to measure the accumulation of the contrast agent in the tumor. Changes in tumor to brain contrast because of active targeting were compared with a nontargeted contrast agent. Effective targeting was confirmed with near infrared measurements and fluorescent microscopic analysis. The results showed that there was a statistically significant (P < .01) difference in normalized T(2) between healthy brain and tumor tissue 10 min, 1 h, and 2 h point postinjection of the anti-IGFBP7 single domain antibody targeted and nontargeted iron oxide nanoparticles. A statistical difference remained in animals treated with targeted nanoparticles 24 h postinjection only. The MRI, near infrared imaging, and fluorescent microscopy studies showed corresponding spatial and temporal changes. We concluded that the developed anti-IGFBP7-iron oxide single domain antibody-targeted MRI contrast agent selectively binds to abnormal vessels within a glioblastoma. T(2)-weighted MRI and near infrared imaging are able to detect the targeting effects in brain tumors.

Background: ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood-brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells., Methods: Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines., Results: Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions., Conclusion: ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.

Molecular imaging enables the non-invasive investigation of cellular and molecular processes. Although there are challenges to overcome, the development of targeted contrast agents to increase the sensitivity of molecular imaging techniques is essential for their clinical translation. In this study, spontaneously forming, small unilamellar vesicles (sULVs) (30 nm diameter) were used as a platform to build a bimodal (i.e., optical and magnetic resonance imaging (MRI)) targeted contrast agent for the molecular imaging of brain tumors. sULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA), functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 single domain), and incorporated a near infrared dye (Cy5.5). The resultant sULVs were characterized in vitro using small angle neutron scattering (SANS), phantom MRI and dynamic light scattering (DLS). Antibody targeted and nontargeted Gd loaded sULVs labeled with Cy5.5 were assessed in vivo in a brain tumor model in mice using time domain optical imaging and MRI. The results demonstrated that a spontaneously forming, nanosized ULVs loaded with a high payload of Gd can selectively target and image, using MR and optical imaging, brain tumor vessels when functionalized with anti-IGFBP7 single domain antibodies. The unique features of these targeted sULVs make them promising molecular MRI contrast agents.

The development of imaging and therapeutic agents against neuronal targets is hampered by the limited access of probes into the central nervous system across the blood-brain barrier (BBB). The evaluation of drug penetration into the brain in experimental models often requires complex procedures, including drug radiolabeling, as well as determinations in multiple animals for each condition or time point. Prospective in vivo imaging of drug biodistribution may provide an alternative to "classical" pharmacokinetics and biodistribution studies in that a contrast-enhanced imaging signal could serve as a surrogate for the amount of drug or biologic delivered to the organ of interest. For the brain-targeting applications, it is necessary to develop formulation strategies that enable a simultaneous drug and contrast agent delivery across the BBB. In this chapter, we describe methods for encapsulating drugs into liposome nanocarriers with surface display of both the imaging contrast agent for one or multiple imaging modalities and the single-domain antibody that undergoes receptor-mediated transcytosis across the BBB. Contrast-enhanced imaging signal detected in the brain after intravenous injection of such formulation(s) is proportional to the amount of drug delivered into the brain parenchyma. This method allows for a prospective, noninvasive estimation of drug delivery, accumulation, and elimination from the brain.

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications., Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy., Results: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels., Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

The aim of the study was to determine the effect of early tumor growth on T(2) relaxation times in an experimental glioma model. A 9.4-T magnetic resonance imaging (MRI) system was used for the investigations. An animal model (n=12) of glioma was established using an intracranial inoculation of U87MGdEGFRvIII cells. The imaging studies were performed from Day 10 through Day 13 following tumor inoculation. Tumor blood vessel density was determined using quantitative immunochemistry. Tumor volume was measured daily using MR images. T(2) values of the tumor were measured in five areas across the tumor and calculated using a single exponential fitting of the echo train. The measurements on Days 10 and 13 after tumor inoculation showed a 20% increase in T(2). The changes in T(2) correlated with the size of the tumor. Statistically significant differences in T(2) values were observed between the edge of the tumor and the brain tissue on Days 11, 12 and 13 (P=.014, .008, .001, respectively), but not on Day 10 (P=.364). The results show that T(2)-weighted MRI may not detect glioma during an early phase of growth. T(2) increases in growing glioma and varies heterogenously across the tumor., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications., Experimental Approach: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies., Key Results: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions., Conclusions: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.