Mary-Ann Elvina Xavier1, Shihui Liu2, Stephen H Leppla3, Bart Cornelissen4. 1. CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom. 2. Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, Pittsburgh, USA. 3. Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA. 4. CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom. Electronic address: bart.cornelissen@oncology.ox.ac.uk.
Abstract
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.
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.
Authors: Bryan A Krantz; Amar D Trivedi; Kristina Cunningham; Kenneth A Christensen; R John Collier Journal: J Mol Biol Date: 2004-11-26 Impact factor: 5.469
Authors: N S Duesbery; C P Webb; S H Leppla; V M Gordon; K R Klimpel; T D Copeland; N G Ahn; M K Oskarsson; K Fukasawa; K D Paull; G F Vande Woude Journal: Science Date: 1998-05-01 Impact factor: 47.728
Authors: Damian Wild; Jörg S Schmitt; Mihaela Ginj; Helmut R Mäcke; Bert F Bernard; Eric Krenning; Marion De Jong; Sandra Wenger; Jean-Claude Reubi Journal: Eur J Nucl Med Mol Imaging Date: 2003-08-21 Impact factor: 9.236
Authors: Nathalie Matusiak; Aren van Waarde; Rainer Bischoff; Ruth Oltenfreiter; Christophe van de Wiele; Rudi A J O Dierckx; Philip H Elsinga Journal: Curr Pharm Des Date: 2013 Impact factor: 3.116
Authors: Andrei P Pomerantsev; Rita M McCall; Margaret Chahoud; Nathan K Hepler; Rasem Fattah; Stephen H Leppla Journal: PLoS One Date: 2017-08-22 Impact factor: 3.240
Authors: Stephanie L Slania; Deepankar Das; Ala Lisok; Yong Du; Zirui Jiang; Ronnie C Mease; Steven P Rowe; Sridhar Nimmagadda; Xing Yang; Martin G Pomper Journal: J Med Chem Date: 2021-03-17 Impact factor: 8.039