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

Abstract

BackgroundPretargeting-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.ResultsBioorthogonal 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (10.1186/s13550-017-0344-6) contains supplementary material, which is available to authorized users.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…Nevertheless, our pretargeted imaging results are more comparable to conventional imaging than previous efforts with HER2, in which tumor contrast was poor or overshadowed by gut clearance. 20,45 Even if an intermediate imaging time point (e.g., 6 hours) had provided marginal improvements in tumor delineation for 7C2-TCO x , perhaps in part due to bladder voiding, the persistence of click reaction (radioimmunoconjugate) products in systemic circulation would have likely overshadowed the relatively low same-day tumor signal.…”

“…11 These IEDDA reactions have second-order rate constants ranging from k 2  = 10 3 –10 6 M −1 s −1 , which are primarily driven by expulsion of N 2 and TCO strain relief following the cycloaddition 12,13 Furthermore, electron withdrawing substituents attached to the tetrazine confer increased reaction rates between the dienophile and diene. 14,15 Successful pretargeted in vivo nuclear imaging has been demonstrated in preclinical models using PET isotopes, including 11 C (t 1/2 20.3 minutes), 16,17 18 F (t 1/2 110 minutes) 18–20 and 64 Cu (t 1/2 12.7 hours), 4,21–24 as well as SPECT isotopes including 99m Tc (t 1/2 6 hours), 25–27 111 In (t 1/2 2.8 days) 28–30 and 177 Lu (t 1/2 6.7 days). 27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups.…”

“…For instance, clinical imaging of human epidermal growth factor receptor 2 (HER2)-expressing tumors has been actively pursued via directly labeled antibodies, 40,41 antibody fragments 42 and affibodies 43 in an effort to improve patient selection and response monitoring for HER2-targeted cancer therapies. 44 Fewer examples exist in which internalizing targets have been pretargeted, 20,39 likely due to risk of time-dependent cell surface depletion of TCO moieties available for click reaction through antibody-bound receptor internalization, thereby decreasing image contrast. For instance, use of enzyme-based approaches for pretargeted imaging as an alternative to IEDDA reactions was successful for a non-internalizing antigen (TAG-72), but not for an internalizing antigen (HER2).…”

“…45 However, there is a recent interest in pretargeted imaging of internalizing targets such as HER2 despite these challenges. 20,39 …”

Temporal Modulation of HER2 Membrane Availability Increases Pertuzumab Uptake and Pretargeted Molecular Imaging of Gastric Tumors

“…The internalization of HER2 to the intracellular compartment not only decreases the ability of 89 Zr-labeled antibodies to target HER2positive tumors but also precludes the use of pretargeted strategies for molecular imaging and therapy (31)(32)(33). Pretargeting approaches have been developed to reduce radiation doses to healthy tissues associated with antibodies radiolabeled with long-lived radionuclides.…”