Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

EJNMMI Research, Dec 2017

Background Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Results Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t ½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. Conclusions We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.

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Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Keinänen et al. EJNMMI Research Pretargeting of internalizing trastuzumab 18 and cetuximab with a F-tetrazine tracer in xenograft models Outi Keinänen 0 1 Kimberly Fung 0 2 6 Jacob Pourat 0 Vilma Jallinoja 0 1 Delphine Vivier 0 2 NagaVara Kishore Pillarsetty 0 4 Anu J. Airaksinen 1 Jason S. Lewis 0 3 4 5 Brian M. Zeglis 0 2 6 Mirkka Sarparanta 0 1 0 Department of Radiology, Memorial Sloan Kettering Cancer Center , 1275 York Avenue, New York, NY 10065 , USA 1 Department of Chemistry , Radiochemistry , University of Helsinki , P.O. Box 55, FI-00014 Helsinki , Finland 2 Department of Chemistry, Hunter College and the Graduate Center of the City University of New York , 695 Park Avenue, New York, NY 10065 , USA 3 Department of Pharmacology, Weill Cornell Medical College , 1300 York Avenue, New York, NY 10065 , USA 4 Department of Radiology, Weill Cornell Medical College , 1300 York Avenue, New York, NY 10065 , USA 5 Program in Molecular Pharmacology, Memorial Sloan Kettering Cancer Center , 1275 York Avenue, New York, NY 10065 , USA 6 Ph.D. Program in Chemistry, Graduate Center of the City University of New York , New York 10016, NY , USA Background: Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Results: Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. Conclusions: We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end. Fluorine-18; Inverse electron-demand Diels-Alder (IEDDA) reaction; Pretargeting; Tetrazine; Trans-cyclooctene Background During recent years, the radiolabelling of clinically relevant monoclonal antibodies (mAbs), like cetuximab and trastuzumab, has been under intense investigation for the generation of diagnostic and therapeutic radiopharmaceuticals that can identify and treat tumours expressing the target antigen [ 1 ]. Traditional radiolabelling methods of mAbs for PET (positron emission tomography) and SPECT (single-photon emission computed tomography) usually involve the use of long-lived radioisotopes, such as zirconium-89 (t½ = 3.3 days), indium-111 (t½ = 2.8 days), and iodine-124 (t½ = 4.2 days); the half-lives of which are matched to the biological half-lives of mAbs. Although the target-to-background ratios obtained with directly radiolabelled mAbs are excellent, typically, several hours to days are required before non-target radioactivity has decreased to sufficiently low levels necessitating the use of long-lived isotopes. An additional caveat is the increased radiation burden to the subject, especially to the bone marrow resulting from long circulation time of mAbs. In pretargeting, the antibody is radiolabelled in vivo after it has accumulated to the tumour and most of the unbound antibody has cleared from the blood flow (Fig. 1), enabling the use of short-lived radioisotopes with dosimetric benefits [ 2–4 ]. Earlier approaches to pretargeting included streptavidin-biotin system [ 5 ], bispecific antibodies targeting the antigen with In-DTPA [ 6 ] or Y-DOTA [ 7 ] complexes, and antibody-modified oligonucleotide conjugates [ 8 ]. In the recent years, bioorthogonal chemical reactions have emerged as efficient tools for pretargeted nuclear imaging of mAbs [ 2, 4, 9–15 ]. Bioorthogonal reactions occur under physiological conditions without interfering with or being interfered (...truncated)


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Outi Keinänen, Kimberly Fung, Jacob Pourat, Vilma Jallinoja, Delphine Vivier, NagaVara Kishore Pillarsetty, Anu J. Airaksinen, Jason S. Lewis, Brian M. Zeglis, Mirkka Sarparanta. Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models, EJNMMI Research, 2017, pp. 95, Volume 7, Issue 1, DOI: 10.1186/s13550-017-0344-6