Fluorophore−Quencher Based Activatable Targeted Optical Probes for Detecting in Vivo Cancer Metastases

Abstract

In vivo molecularly targeted fluorescence imaging of tumors has been proposed as a strategy for improving cancer detection and management. Activatable fluorophores, which increased their fluorescence by 10-fold after binding tumor cells, result in much higher target to background ratios than conventional fluorophores. We developed an in vivo targeted activatable optical imaging probe based on a fluorophore-quencher pair, bound to a targeting moiety. With this system, fluorescence is quenched by the fluorophore-quencher interaction outside cancer cells, but is activated within the target cells by dissociation of the fluorophore-quencher pair. We selected the TAMRA (fluorophore)-QSY7 (quencher) pair and conjugated it to either avidin (targeting the D-galactose receptor) or trastuzumab (a monoclonal antibody against the human epithelial growth factor receptor type2 (HER2/neu)) and evaluated their performance in mouse models of cancer. Two probes, TAMRA-QSY7 conjugated avidin (Av-TM-Q7) and trastuzumab (Traz-TM-Q7) were synthesized. Both demonstrated better than similar self-quenching probes. In vitro fluorescence microscopic studies of SHIN3 and NIH/3T3/HER2+ cells demonstrated that Av-TM-Q7 and Traz-TM-Q7 produced high intracellular fluorescent signal. In vivo imaging with Av-TM-Q7 and Traz-TM-Q7 in mice enabled the detection of small tumors. This molecular imaging probe, based on a fluorophore-quencher pair conjugated to a targeting ligand, successfully detected tumors in vivo due to its high activation ratio and low background signal. Thus, these activatable probes, based on the fluorophore-quencher system, hold promise clinically for "see and treat" strategies of cancer management.

An Antibody–Drug Conjugate That Targets Tissue Factor Exhibits Potent Therapeutic Activity against a Broad Range of Solid Tumors

“…Goat-anti-human IgG Fab-fragments (Jackson Immunoresearch Laboratories) were conjugated with the fluorophore and quencher pair TAMRA/QSY7 (Fab-TAMRA/QSY7) as described (24). Tissue factor HuMab (1 mg/mL) was preincubated with Fab-TAMRA/QSY7 (2 mg/mL; 30 minutes, 4 C), and the complex was added to SK-OV-3 or A431 cells while shaking (200 rpm, 37 C).…”

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

“…In the context of molecular imaging, a variety of such probes have been developed (14)(15)(16)(17)(18)(19)(20)(21). Typical mechanisms include enzymatic cleavage of a quencher group from the fluorescence moiety using cancer-related enzymes (15)(16)(17)(18), intracellular degradation of fluorescence-quenched protein against cancer cell receptors (19,20), and surrounding-sensitized fluorescence labels conjugated to macromolecular ligands selectively endocytosed by cancer cells (8).…”

“…Typical mechanisms include enzymatic cleavage of a quencher group from the fluorescence moiety using cancer-related enzymes (15)(16)(17)(18), intracellular degradation of fluorescence-quenched protein against cancer cell receptors (19,20), and surrounding-sensitized fluorescence labels conjugated to macromolecular ligands selectively endocytosed by cancer cells (8). However, these mechanisms cannot be utilized to develop aptamer-based activatable probes.…”

High-sensitivity detection of breast tumorsin vivoby use of a pH-sensitive near-infrared fluorescence probe

“…Other groups like Urano et al 9 and Ogawa et al 41 have also achieved high tumor-to-background ratios with pH-sensitive or pH-activatable Herceptin conjugates utilizing photon-induced electron transfer (PeT)-operated fluorescent reporters and a TAMRA-QSY7 fluorophore-quencher pair conjugated to Herceptin. However, as these probes both emit in the visible wavelength region, the limited tissue penetration hampered the generation of in vivo data and allowed only ex vivo measurements of the opened abdominal cavity of mice to detect fluorescent signals.…”