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 (EG2) and two higher valency format constructs, bivalent EG2-hFc and pentavalent V2C-EG2 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 EG2 to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10-to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG2 and EG2-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG2, V2C-EG2 and EG2-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG2-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 EG2-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.

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.

Ag presentation to CD8+ T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 × 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8+ T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8+ T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8+ T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intracellular proliferation of virulent pathogens may be an important mechanism of immune evasion.

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 (KD=40–50 n) 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.