Pre-labelling versus direct labelling of anthrax proteins for imaging of matrix metalloproteinases activity using DOTA-GA.
Xavier M-AE., Liu S., Leppla SH., Cornelissen B.
INTRODUCTION: Increased activity of matrix metalloproteases (MMPs) is associated with reduced survival in several cancer subtypes. Aiming to produce an MMP tumour cell-selective cytotoxin, we genetically modified both components of the AB-type lethal toxin from Bacillus anthracis. Component A, Protective Antigen (PA-WT), was re-engineered to form an oligomeric pore in cell membranes only when cleaved by MMPs (PA-L1). The pore-translocation domain (LFn - N-terminal, 30 kDa) of the Lethal Factor (LF), component B, was fused to the catalytic domain of Pseudomonas exotoxin-A to increase its cytotoxic effect when delivered to cancerous cells. Here, we develop radiolabelled forms of LFn for MMP activity imaging by SPECT using the LFn/PA-L1 system. METHODS: DOTA-GA-maleimide was conjugated to LFn to allow radiolabelling with 111In via two different routes: (1) LFn was conjugated with maleimide-DOTA-GA under mild conditions, and then radiolabelled in acidic conditions at 95°C, or (2) 111In was coordinated to maleimide-DOTA-GA first and then conjugated via maleimide chemistry to LFn. Circular Dichroism Spectroscopy of LFn was performed to evaluate changes in its secondary structure. Cell uptake assays using the differently labelled forms of [111In]In-DOTA-GA-LFn in the presence or not of PA-WT or PA-L1 were performed. RESULTS: LFn was successfully radiolabelled by either strategy. Comparison of the secondary structure content of LFn exposed to 37°C or 95°C, showed a loss of alpha helix content at higher temperatures. Cell uptake of both forms of [111In]In-DOTA-GA-LFn, labelled directly or indirectly, was significantly higher in MMP-positive cells, in the presence of PA-L1, compared to controls. Notably, despite being exposed to high temperatures, uptake of directly labelled [111In]In-DOTA-GA-LFndir was higher than indirectly labelled [111In]In-DOTA-GA-LFnindir. CONCLUSIONS: 111In-radiolabelling of LFn results in a functional molecule that targets MMP-activity in cells when combined with PA-L1. [111In]In-LFn/PA-L1 is a promising MMP activity imaging agent for SPECT imaging.