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Licensed Unlicensed Requires Authentication Published by De Gruyter September 21, 2018

Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics

  • Gezim Bala , Maxine Crauwels , Anneleen Blykers , Isabel Remory , Andrea L.J. Marschall , Stefan Dübel , Laurent Dumas , Alexis Broisat , Charlotte Martin , Steven Ballet , Bernard Cosyns , Vicky Caveliers , Nick Devoogdt , Catarina Xavier and Sophie Hernot ORCID logo EMAIL logo
From the journal Biological Chemistry


Radiolabeling of nanobodies with radiometals by chelation has the advantage of being simple, fast and easy to implement in clinical routine. In this study, we validated 68Ga/111In-labeled anti-VCAM-1 nanobodies as potential radiometal-based tracers for molecular imaging of atherosclerosis. Both showed specific targeting of atherosclerotic lesions in ApoE−/− mice. Nevertheless, uptake in lesions and constitutively VCAM-1 expressing organs was lower than previously reported for the 99mTc-labeled analog. We further investigated the impact of different radiolabeling strategies on the in vivo biodistribution of nanobody-based tracers. Comparison of the pharmacokinetics between 68Ga-, 18F-, 111In- and 99mTc-labeled anti-VCAM-1 nanobodies showed highest specific uptake for 99mTc-nanobody at all time-points, followed by the 68Ga-, 111In- and 18F-labeled tracer. No correlation was found with the estimated number of radioisotopes per nanobody, and mimicking specific activity of other radiolabeling methods did not result in an analogous biodistribution. We also demonstrated specificity of the tracer using mice with a VCAM-1 knocked-down phenotype, while showing for the first time the in vivo visualization of a protein knock-down using intrabodies. Conclusively, the chosen radiochemistry does have an important impact on the biodistribution of nanobodies, in particular on the specific targeting, but differences are not purely due to the tracer’s specific activity.


This work was supported by the Research Foundation-Flanders, Belgium, Funder Id: 10.13039/501100003130 (FWO G066615N and G005815N), the Strategic Research Program-Growth funding of the Vrije Universiteit Brussel (VUB) and the Wetenschappelijk Fonds Willy Gepts. We thank Cindy Peleman and Jan De Jonge for their technical assistance.


Andrews, J.P.M., Fayad, Z.A., and Dweck, M.R. (2018). New methods to image unstable atherosclerotic plaques. Atherosclerosis 272, 118–128.10.1016/j.atherosclerosis.2018.03.021Search in Google Scholar PubMed PubMed Central

Bala, G., Blykers, A., Xavier, C., Descamps, B., Broisat, A., Ghezzi, C., Fagret, D., Van Camp, G., Caveliers, V., Vanhove, C., et al. (2016). Targeting of vascular cell adhesion molecule-1 by 18F-labeled nanobodies for PET/CT imaging of inflamed atherosclerotic plaques. Eur. Heart J. Cardiovasc. Imaging 17, 1001–1008.10.1093/ehjci/jev346Search in Google Scholar PubMed

Bala, G., Baudhuin, H., Remory, I., Gillis, K., Debie, P., Krasniqi, A., Lahoutte, T., Raes, G., Devoogdt, N., Cosyns, B., et al. (2018a). Evaluation of 99mTc-radiolabeled macrophage-mannose receptor-specific nanobodies for targeting of atherosclerotic lesions in mice. Mol. Imaging Biol. 20, 260–267.10.1007/s11307-017-1117-3Search in Google Scholar PubMed

Bala, G., Broisat, A., Lahoutte, T., and Hernot, S. (2018b). Translating molecular imaging of the vulnerable plaque-a vulnerable project? Mol. Imaging Biol. 20, 337–339.10.1007/s11307-017-1147-xSearch in Google Scholar PubMed

Blykers, A., Schoonooghe, S., Xavier, C., D’Hoe, K., Laoui, D., D’Huyvetter, M., Vaneycken, I., Cleeren, F., Bormans, G., Heemskerk, J., et al. (2015). PET imaging of macrophage mannose receptor-expressing macrophages in tumor stroma using 18F-radiolabeled camelid single-domain antibody fragments. J. Nucl. Med. 56, 1265–1271.10.2967/jnumed.115.156828Search in Google Scholar PubMed

Brasse, D. and Nonat, A. (2015). Radiometals: towards a new success story in nuclear imaging? Dalton Trans. 44, 4845–4858.10.1039/C4DT02911ASearch in Google Scholar

Broisat, A., Hernot, S., Toczek, J., De Vos, J., Riou, L.M., Martin, S., Ahmadi, M., Thielens, N., Wernery, U., Caveliers, V., et al. (2012). Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ. Res. 110, 927–937.10.1161/CIRCRESAHA.112.265140Search in Google Scholar PubMed PubMed Central

Broisat, A., Toczek, J., Dumas, L.S., Ahmadi, M., Bacot, S., Perret, P., Slimani, L., Barone-Rochette, G., Soubies, A., Devoogdt, N., et al. (2014). 99mTc-cAbVCAM1-5 imaging is a sensitive and reproducible tool for the detection of inflamed atherosclerotic lesions in mice. J. Nucl. Med. 55, 1678–1684.10.2967/jnumed.114.143792Search in Google Scholar PubMed

Chakravarty, R., Goel, S., and Cai, W. (2014). Nanobody: the “magic bullet” for molecular imaging? Theranostics 4, 386–398.10.7150/thno.8006Search in Google Scholar PubMed PubMed Central

D’Huyvetter, M., De Vos, J., Xavier, C., Pruszynski, M., Sterckx, Y.G.J., Massa, S., Raes, G., Caveliers, V., Zalutsky, M., Lahoutte, T., et al. (2017). 131I-labeled anti-HER2 camelid sdAb as a theranostic tool in cancer treatment. Clin. Cancer Res. 23, 6616–6628.10.1158/1078-0432.CCR-17-0310Search in Google Scholar PubMed PubMed Central

Debie, P., Van Quathem, J., Hansen, I., Bala, G., Massa, S., Devoogdt, N., Xavier, C., and Hernot, S. (2017). Effect of dye and conjugation chemistry on the biodistribution profile of near-infrared-labeled nanobodies as tracers for image-guided surgery. Mol. Pharm. 14, 1145–1153.10.1021/acs.molpharmaceut.6b01053Search in Google Scholar PubMed

Debie, P., Vanhoeij, M., Poortmans, N., Puttemans, J., Gillis, K., Devoogdt, N., Lahoutte, T., and Hernot, S. (2018). improved debulking of peritoneal tumor implants by near-infrared fluorescent nanobody image guidance in an experimental mouse model. Mol. Imaging Biol. 20, 361–367.10.1007/s11307-017-1134-2Search in Google Scholar PubMed

Dumas, L.S., Briand, F., Clerc, R., Brousseau, E., Montemagno, C., Ahmadi, M., Bacot, S., Soubies, A., Perret, P., Riou, L.M., et al. (2017). Evaluation of antiatherogenic properties of ezetimibe using 3H-labeled low-density-lipoprotein cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE−/− mice fed the paigen diet. J. Nucl. Med. 58, 1088–1093.10.2967/jnumed.116.177279Search in Google Scholar PubMed

Germano, G., Berman, D.S., and Slomka, P. (2016). Technical aspects of cardiac PET imaging and recent advances. Cardiol. Clin. 34, 13–23.10.1016/j.ccl.2015.07.015Search in Google Scholar PubMed

Goethals, L.R., Weytjens, C.D., De Geeter, F., Droogmans, S., Caveliers, V., Keyaerts, M., Vanhove, C., Van Camp, G., Bossuyt, A., and Lahoutte, T. (2009). Regional quantitative analysis of small animal myocardial sympathetic innervation and initial application in streptozotocin induced diabetes. Contrast Media. Mol. Imaging 4, 174–182.10.1002/cmmi.278Search in Google Scholar PubMed

Hernot, S., Unnikrishnan, S., Du, Z., Shevchenko, T., Cosyns, B., Broisat, A., Toczek, J., Caveliers, V., Muyldermans, S., Lahoutte, T., et al. (2012). Nanobody-coupled microbubbles as novel molecular tracer. J. Control Release 158, 346–353.10.1016/j.jconrel.2011.12.007Search in Google Scholar PubMed PubMed Central

Jacobson, O., Kiesewetter, D.O., and Chen, X. (2014). Fluorine-18 radiochemistry, labeling strategies and synthetic routes. Bioconjug. Chem. 26, 1–18.10.1021/bc500475eSearch in Google Scholar PubMed PubMed Central

Keyaerts, M., Xavier, C., Heemskerk, J., Devoogdt, N., Everaert, H., Ackaert, C., Vanhoeij, M., Duhoux, F.P., Gevaert, T., Simon, P., et al. (2016). Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. J. Nucl. Med. 57, 27–33.10.2967/jnumed.115.162024Search in Google Scholar PubMed

Konijnenberg, M.W., Breeman, W.A., de Blois, E., Chan, H.S., Boerman, O.C., Laverman, P., Kolenc-Peitl, P., Melis, M., and de Jong, M. (2014). Therapeutic application of CCK2R-targeting PP-F11: influence of particle range, activity and peptide amount. EJNMMI Res. 4, 47.10.1186/s13550-014-0047-1Search in Google Scholar PubMed PubMed Central

Krasniqi, A., D’Huyvetter, M., Xavier, C., Van der Jeught, K., Muyldermans, S., Van Der Heyden, J., Lahoutte, T., Tavernier, J., and Devoogdt, N. (2017). Theranostic radiolabeled anti-CD20 sdAb for targeted radionuclide therapy of non-Hodgkin lymphoma. Mol. Cancer Ther. 16, 2828–2839.10.1158/1535-7163.MCT-17-0554Search in Google Scholar PubMed

Krasniqi, A., D’Huyvetter, M., Devoogdt, N., Frejd, F.Y., Sorensen, J., Orlova, A., Keyaerts, M., and Tolmachev, V. (2018). Same-day imaging using small proteins: clinical experience and translational prospects in oncology. J. Nucl. Med. 59, 885–891.10.2967/jnumed.117.199901Search in Google Scholar PubMed

Krishnan, H.S., Ma, L., Vasdev, N., and Liang, S.H. (2017). 18F-Labeling of sensitive biomolecules for positron emission tomography. Chemistry 23, 15553–15577.10.1002/chem.201701581Search in Google Scholar PubMed PubMed Central

Kung, M.P. and Kung, H.F. (2005). Mass effect of injected dose in small rodent imaging by SPECT and PET. Nucl. Med. Biol. 32, 673–678.10.1016/j.nucmedbio.2005.04.002Search in Google Scholar PubMed

Marschall, A.L., Single, F.N., Schlarmann, K., Bosio, A., Strebe, N., van den Heuvel, J., Frenzel, A., and Dubel, S. (2014). Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies. mAbs 6, 1394–1401.10.4161/mabs.34377Search in Google Scholar PubMed PubMed Central

Martiniova, L., Palatis, L., Etchebehere, E., and Ravizzini, G. (2016). Gallium-68 in medical imaging. Curr. Radiopharm. 9, 187–207.10.2174/1874471009666161028150654Search in Google Scholar PubMed

Massa, S., Vikani, N., Betti, C., Ballet, S., Vanderhaegen, S., Steyaert, J., Descamps, B., Vanhove, C., Bunschoten, A., van Leeuwen, F.W., et al. (2016). Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities. Contrast. Media Mol. Imaging. 11, 328–339.10.1002/cmmi.1696Search in Google Scholar PubMed

Naghavi, M., Libby, P., Falk, E., Casscells, S.W., Litovsky, S., Rumberger, J., Badimon, J.J., Stefanadis, C., Moreno, P., Pasterkamp, G., et al. (2003). From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part I. Circulation 108, 1664–1672.10.1161/01.CIR.0000087480.94275.97Search in Google Scholar PubMed

Notni, J., Steiger, K., Hoffmann, F., Reich, D., Schwaiger, M., Kessler, H., and Wester, H.J. (2016). Variation of specific activities of 68Ga-aquibeprin and 68Ga-avebetrin enables selective PET imaging of different expression levels of integrins 5 1 and v 3. J. Nucl. Med. 57, 1618–1624.10.2967/jnumed.116.173948Search in Google Scholar PubMed

Price, E.W. and Orvig, C. (2014). Matching chelators to radiometals for radiopharmaceuticals. Chem. Soc. Rev. 43, 260–290.10.1039/C3CS60304KSearch in Google Scholar

Quillard, T. and Libby, P. (2012). Molecular imaging of atherosclerosis for improving diagnostic and therapeutic development. Circ. Res. 111, 231–244.10.1161/CIRCRESAHA.112.268144Search in Google Scholar PubMed PubMed Central

Schroeder, R.P., De Blois, E., De Ridder, C.M., Van Weerden, W.M., Breeman, W.A., and de Jong, M. (2012). Improving radiopeptide pharmacokinetics by adjusting experimental conditions for bombesin receptor-targeted imaging of prostate cancer. Q. J. Nucl. Med. Mol. Imaging 56, 468–475.Search in Google Scholar

Steeland, S., Vandenbroucke, R.E., and Libert, C. (2016). Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov. Today 21, 1076–1113.10.1016/j.drudis.2016.04.003Search in Google Scholar PubMed

Tarkin, J.M., Joshi, F.R., and Rudd, J.H. (2014). PET imaging of inflammation in atherosclerosis. Nat. Rev. Cardiol. 11, 443–457.10.1038/nrcardio.2014.80Search in Google Scholar PubMed

Tolmachev, V., Rosik, D., Wallberg, H., Sjoberg, A., Sandstrom, M., Hansson, M., Wennborg, A., and Orlova, A. (2010). Imaging of EGFR expression in murine xenografts using site-specifically labeled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount. Eur. J. Nucl. Med. Mol. Imaging 37, 613–622.10.1007/s00259-009-1283-xSearch in Google Scholar PubMed

Tolmachev, V., Wallberg, H., Sandstrom, M., Hansson, M., Wennborg, A., and Orlova, A. (2011). Optimal specific radioactivity of anti-HER2 Affibody molecules enables discrimination between xenografts with high and low HER2 expression levels. Eur. J. Nucl. Med. Mol. Imaging 38, 531–539.10.1007/s00259-010-1646-3Search in Google Scholar PubMed

Vandesquille, M., Li, T., Po, C., Ganneau, C., Lenormand, P., Dudeffant, C., Czech, C., Grueninger, F., Duyckaerts, C., Delatour, B., et al. (2017). Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer’s disease. mAbs 9, 1016–1027.10.1080/19420862.2017.1342914Search in Google Scholar PubMed PubMed Central

Vaneycken, I., Devoogdt, N., Van Gassen, N., Vincke, C., Xavier, C., Wernery, U., Muyldermans, S., Lahoutte, T., and Caveliers, V. (2011). Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J. 25, 2433–2446.10.1096/fj.10-180331Search in Google Scholar PubMed

Velikyan, I. (2013). Prospective of 68Ga-radiopharmaceutical development. Theranostics 4, 47–80.10.7150/thno.7447Search in Google Scholar PubMed PubMed Central

Xavier, C., Devoogdt, N., Hernot, S., Vaneycken, I., D’Huyvetter, M., De Vos, J., Massa, S., Lahoutte, T., and Caveliers, V. (2012). Site-specific labeling of his-tagged Nanobodies with 99mTc: a practical guide. Methods Mol. Biol. 911, 485–490.10.1007/978-1-61779-968-6_30Search in Google Scholar PubMed

Xavier, C., Vaneycken, I., D’Huyvetter, M., Heemskerk, J., Keyaerts, M., Vincke, C., Devoogdt, N., Muyldermans, S., Lahoutte, T., and Caveliers, V. (2013). Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 nanobodies for iPET imaging of HER2 receptor expression in cancer. J. Nucl. Med. 54, 776–784.10.2967/jnumed.112.111021Search in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (

Received: 2018-07-30
Accepted: 2018-08-20
Published Online: 2018-09-21
Published in Print: 2019-02-25

©2019 Walter de Gruyter GmbH, Berlin/Boston

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