B. anthracis protective antigen: a molecular imaging agent targeting squamous cell carcinoma

Abstract

Targeted molecular imaging is an emerging non-invasive tool for the identification and characterisation of cancers based on physiological and molecular properties. Molecular imaging is likely to play a significant role in the development and validation of future biomarkers due to the ability to identify and monitor real time tumour characteristics noninvasively. There is currently a need to identify novel biomarkers that can be used to target, or inform on, tumour specific processes. The human anthrax toxin receptors Tumour Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2) have been identified as potential novel tumour biomarkers. TEM8 and CMG2 are cell surface receptors involved in angiogenesis and extracellular matrix homeostasis, processes that are important for tumour growth and metastasis. TEM8 is particularly interesting due to its tumour specific upregulation and undetectable expression in many healthy adult tissues. One method to target these receptors is to exploit their exogenous ligand Protective Antigen (PA). PA is a protein produced by the bacterium B. anthracis that mediates binding and internalisation of toxins into cells. Whilst preclinical studies have shown that PA can be used to specifically target toxins to tumours, the tumour specific localisation of PA in vivo has yet to be demonstrated. We have expressed recombinant PA, using an E. coli expression system, and purified the protein by liquid chromatography techniques. PA was non-specifically labelled with an amine reactive near infra-red fluorescent dye for optical imaging or 89 Zr-Desferoxamine (89 Zr-DFO) for positron emission tomography (PET). The labelled PA was intravenously (IV) injected into female HPV38E6E7-FVB mice, a transgenic mouse model of squamous cell carcinoma (SCC) that endogenously produces tumours in response to repeated UV irradiation. The fluorescently labelled PA protein was highly specific to the SCC tumours, resulting in rapid tumour uptake and exclusive tumour identification 24 hours post injection. Post mortem analysis, 48 hours post injection, showed that the fluorescence in the tumour was significantly higher (p < 0.002, two tailed paired T test) than the adjacent skin, the liver, and the kidneys. Immunofluorescence analysis of formalin-fixed paraffin-embedded skin Publications included in this thesis No publications included.