Related Experiment Video
Updated: Jun 13, 2026
An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
Published on: June 6, 2022
Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein
Florian Kampmeier1, Judith Niesen, Alexander Koers
1Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany.
Purpose:
The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression.
Methods:
Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model.
Results:
The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection.
Conclusion:
The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.
Insights
A novel imaging probe targets epidermal growth factor receptor (EGFR) in tumors, showing rapid accumulation and clearance for better imaging. This tool enables non-invasive assessment of EGFR expression in cancer.
Area of Science:
- Oncology
- Molecular Imaging
- Biotechnology
Background:
- Epidermal growth factor receptor (EGFR) is overexpressed in various cancers, making it a key target for cancer therapy.
- Inhibition of EGFR has shown efficacy in slowing tumor progression.
- Developing effective imaging probes is crucial for non-invasive assessment of EGFR expression in tumors.
Purpose of the Study:
- To design an EGFR-specific imaging probe for enhanced tumor targeting.
- To achieve rapid systemic clearance of the probe for improved imaging quality.
- To facilitate non-invasive assessment of EGFR expression in cancer.
Main Methods:
- Genetic fusion of a single-chain antibody fragment (scFv) with SNAP-tag to create a 48-kDa antibody derivative.
- Site-specific covalent labeling of the fusion protein (425(scFv)SNAP) with a near-infrared (NIR) dye (BG-747).
- Monitoring probe accumulation, specificity, and kinetics using NIR fluorescence imaging in a pancreatic cancer xenograft model.
Main Results:
- The 425(scFv)SNAP fusion protein demonstrated rapid and specific accumulation in tumors.
- High tumor-to-background ratios (TBR) of 33.2 ± 6.3 were achieved 10 hours post-injection due to efficient renal clearance.
- The probe showed efficient competition with unlabeled probe and superior TBR compared to full-length antibodies, with complete clearance by 72 hours.
Conclusions:
- The 425(scFv)SNAP fusion protein effectively targets EGFR-positive tumors with rapid clearance.
- The versatile labeling technique allows attachment of fluorophores for optical imaging.
- This approach is adaptable for developing probes for nuclear imaging by coupling chelating agents.
