A Fluorescent Imaging Probe Based on a Macrocyclic Scaffold That Binds to Cellular EpCAM

Area of Science:

• Biotechnology
• Molecular Imaging
• Biochemistry

Background:

• Epithelial cell adhesion molecule (EpCAM) is a biomarker overexpressed in various carcinomas.
• Current methods for EpCAM detection, such as monoclonal antibody (mAb) staining, face limitations in high cell-density conditions.
• Development of novel, high-affinity probes is crucial for accurate cancer cell imaging and targeted therapies.

Purpose of the Study:

• To design and characterize a novel macrocyclic peptide-based fluorescent probe for specific EpCAM detection.
• To evaluate the probe's binding affinity and performance in visualizing EpCAM-expressing cells, particularly under challenging high cell-density conditions.
• To explore the potential of this molecular probe as an imaging tool and a drug delivery vehicle.

Main Methods:

• Discovery of a 14-mer macrocyclic peptide using the random non-standard peptide-integrated discovery system.
• Characterization of the peptide's binding affinity to the extracellular domain of EpCAM using dissociation constant measurements.
• Assessment of the fluorescent probe's performance in staining EpCAM-expressing MCF7 cells, including under high cell-density conditions.

Main Results:

• A macrocyclic peptide probe with specific binding to EpCAM was identified, exhibiting a low nanomolar dissociation constant (1.7 nM).
• The fluorescently tagged probe, with a molecular weight under 3000 Da, successfully visualized nearly all live cells even in high-density cultures.
• The probe demonstrated superior performance compared to conventional monoclonal antibody staining methods in dense cell populations.

Conclusions:

• The developed macrocyclic peptide probe is a highly specific and sensitive tool for EpCAM detection.
• Its compact size and high affinity enable effective cell visualization, outperforming traditional methods in dense cultures.
• This molecular probe holds significant promise as an advanced imaging agent for EpCAM biomarkers and as a platform for targeted drug conjugate delivery.