Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein.
OBJECTIVE
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.


CONCLUSIONS
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.
Kampmeier
Florian
F
Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany.
Niesen
Judith
J
Koers
Alexander
A
Ribbert
Markus
M
Brecht
Andreas
A
Fischer
Rainer
R
Kiessling
Fabian
F
Barth
Stefan
S
Thepen
Theo
T
D019883:Alkyl and Aryl Transferases / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D004396:Coloring Agents / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D055620:Optical Phenomena; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000378:metabolism*; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013997:Time Factors
D004396:Coloring Agents; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase; D066246:ErbB Receptors
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