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SPECT Imaging with 99mTc-Labeled EGFR-Specific Nanobody for In Vivo Monitoring of EGFR Expression

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Abstract

Purpose

Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody®, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice.

Methods

Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with 99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of 99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR.

Results

FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of 99mTc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6 ± 0.9%IA) compared to total liver uptake (18.9 ± 0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2 ± 0.5%IA/cm3) of 99mTc-8B6 Nanobody compared to DU145 xenografts (1.8 ± 0.3%IA/cm3, p < 0.001).

Conclusion

The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with 99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

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Acknowledgments

L.H. is supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). L.O.T.G. is a Ph.D. fellow of the Horizontal Onderzoek Axis (HOA) of the VUB. T.L. is a Senior Clinical Investigator of the Research Foundation–Flanders (Belgium) (FWO). The research at ICMI is funded by the Interuniversity Attraction Poles Programme–Belgian State–Belgian Science Policy.

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Authors and Affiliations

  1. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Lieven Huang & Patrick De Baetselier

  2. Department of Molecular and Cellular Interactions, VIB, Brussels, Belgium

    Lieven Huang & Patrick De Baetselier

  3. Department of Molecular Biology, Ghent University, Ghent, Belgium

    Lieven Huang

  4. Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Lieven Huang

  5. Laboratory of In Vivo Cellular and Molecular Imaging (ICMI), Department of Nuclear Medicine, University Hospital Vrije Universiteit Brussel (UZ Brussel), Brussels, Belgium

    Lea Olive Tchouate Gainkam, Vicky Caveliers, Chris Vanhove, Marleen Keyaerts, Axel Bossuyt & Tony Lahoutte

  6. Ablynx N.V., Zwijnaarde, Belgium

    Hilde Revets

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  1. Lieven Huang
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Huang, L., Gainkam, L.O.T., Caveliers, V. et al. SPECT Imaging with 99mTc-Labeled EGFR-Specific Nanobody for In Vivo Monitoring of EGFR Expression. Mol Imaging Biol 10, 167–175 (2008). https://doi.org/10.1007/s11307-008-0133-8

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  • DOI: https://doi.org/10.1007/s11307-008-0133-8

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