Abstract 4888: Development of a novel FAST cathepsin-activatable NIR agent for tumor imaging
Authors: Kossodo Sylvie C. , Morin Jeffrey , Delaney Jeannine , Cuneo Garry , Handy Emma , Groves Kevin , Zhang Jun , Rajopadhye Milind , Yared Wael , Peterson Jeffrey D. 
Published: June 2012

Abstract
Fluorescence molecular tomography (FMT) and near-infrared (NIR) imaging agents enable the quantification of kinetic changes in tumor biology in vivo, information useful for the detection, staging, and monitoring of chemotherapeutic efficacy. In particular, imaging agents that become fluorescent upon protease activation in vivo have emerged as valuable tools in oncology research due to the important role of proteases in tumor progression and invasion. Our objective was to develop a novel pan-cathepsin agent with the capability of fast activation kinetics and the potential for early imaging time points in monitoring tumor progression in vivo. To develop this agent, we compared it to an established and well characterized pan-cathepsin activatable imaging agent (ProSenseTM750, a high molecular weight, PEG-decorated NIR-labeled polylysine), which has a long circulation half-life of approximately 20h and offers a long and stable imaging window from 24 to 48h post-injection. ProSenseTM750 FAST is a newly developed 22 kDa molecule based on a novel scaffold consisting of a discrete pair of fluorophores separated by a cleavable sequence and attached to a small pharmacokinetic modifier. To compare selectivity profiles, the agents were assessed for activation by a variety of proteases, including cathepsins and matrix metalloproteases (MMPs). Based on their profiles, both agents show broad pan-cathepsin activation, cleaved by cathepsins B, S, L, and K, with little or no cleavage by MMPs. Consistent with its small architecture, ProSense750 FAST is efficiently activated, approximately 5X faster than the larger ProSense750, offering protease assay readouts as early as 1-2h. In mouse macrophages and 4T1 mouse breast adenocarcinoma cells, both agents were effectively up-taken and activated within the lysosomes for fluorescence microscopy assessment. Nearly complete inhibition of agent activation was achieved using the cell-permeable calpain/cysteine inhibitor E64d. In vivo, FMT imaging of ProSense750 FAST in a 4T-1 tumor model revealed clearly detectable tumor fluorescence as early as 2h after injection, reflecting its short pharmacokinetics (t1/2 ∼ 30 min) and quick in vitro activation profile, with quantification of peak signal around 6h and wash out by 72h. Animals can be optimally imaged at 6 or 24h + 1h, requiring scheduled injection/imaging times. The larger ProSense750 agent, in contrast, shows later optimal imaging (24-48h) but allows greater flexibility in imaging times for large animal cohorts (+ 3h). Careful assessment of organ biodistribution in 4T1 tumor-bearing mice showed different metabolic routes for the two agents, with ProSense750 cleared predominantly through the liver and ProSense750 FAST by renal clearance. These agents offer powerful tools for predicting and monitoring changes in tumor biology in preclinical models of disease.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2011-4888