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Inert coupling of IRDye800CW and zirconium-89 to monoclonal antibodies for single- or dual-mode fluorescence and PET imaging

Nature Protocols volume 8, pages 1010–1018 (2013)Cite this article

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Abstract

IRDye800CW and zirconium-89 (89Zr) have very attractive properties for optical imaging and positron emission tomography (PET) imaging, respectively. Here we describe a procedure for dual labeling of mAbs with IRDye800CW and 89Zr in a current good manufacturing practice (cGMP)-compliant way. IRDye800CW and 89Zr are coupled inertly, without impairment of immunoreactivity and pharmacokinetics of the mAb. Organ and whole-body distribution of the final product can be assessed by optical and PET imaging, respectively. For this purpose, a minimal amount of the chelate N-succinyldesferrioxamine (N-sucDf) is first conjugated to the mAb. Next, N-sucDf-mAb is conjugated with IRDye800CW, after which the N-sucDf-mAb-IRDye800CW is labeled with 89Zr. After each of these three steps, the product is purified by gel filtration. The sequence of this process avoids unnecessary radiation exposure to personnel and takes about 5 h. The process can be scaled up by the production of large batches of premodified mAbs that can be dispensed and stored until they are labeled with 89Zr.

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Figure 1: Schematic overview of the preparation of 89Zr-mAb-IRDye800CW conjugate.
Figure 2: Biodistribution of intravenously injected 89Zr-cetuximab-IRDye800CW (0, 0.5, 1.0 and 2.0 equivalents of IRDye800CW) in A431 xenograft-bearing nude mice at 24 h after injection (percentage of injected dose per gram of tissue ± s.e.m.; n = 5).
Figure 3: Dual-mode imaging of intravenously injected 89Zr-bevacizumab-IRDye800CW (1.0 eq) in a FaDu xenograft-bearing nude mouse at 24 h after injection.
Figure 4: Typical quality-control protein HPLC chromatogram of 89Zr-mAb-IRDye800CW.

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Acknowledgements

This project was financially supported by the Center for Translational Molecular Medicine, project AIRFORCE number 030-103 and by the European Union FP7, ADAMANT. The publication reflects only the authors' views. The European Commission is not liable for any use that may be made of the information contained herein.

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Authors and Affiliations

  1. Department of Otolaryngology/Head and Neck Surgery, Vrije Universiteit (VU) University Medical Center, Amsterdam, The Netherlands

    Ruth Cohen, Danielle J Vugts, Marijke Stigter-van Walsum & Guus A M S van Dongen

  2. Department of Nuclear Medicine and PET Research, VU University Medical Center, Amsterdam, The Netherlands

    Danielle J Vugts, Gerard W M Visser & Guus A M S van Dongen

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  1. Ruth Cohen
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  2. Danielle J Vugts
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  5. Guus A M S van Dongen
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Contributions

All authors contributed extensively to the work presented in the paper. R.C., D.J.V., G.W.M.V. and G.A.M.S.v.D. designed the chemical procedures and experiments. R.C. and M.S-.v.W. did all the chemistry, quality analyses and animal experiments that were used as input data for the protocol. Moreover, they also did the administrative work. D.J.V., G.W.M.V. and G.A.M.S.v.D. gave technical support and conceptual advice. R.C., D.J.V., G.W.M.V. and G.A.M.S.v.D. wrote the protocol.

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Correspondence to Guus A M S van Dongen.

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Cohen, R., Vugts, D., Stigter-van Walsum, M. et al. Inert coupling of IRDye800CW and zirconium-89 to monoclonal antibodies for single- or dual-mode fluorescence and PET imaging. Nat Protoc 8, 1010–1018 (2013). https://doi.org/10.1038/nprot.2013.054

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