Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [⁸⁹Zr]Zr-DFO-PEG₅-Tz

Abstract

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89 Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels−Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t 1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [ 89 Zr]Zr-DFO-PEG 5 -Tz ([ 89 Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89 Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Toward Optimized 89Zr-Immuno-PET: Side-by-Side Comparison of [89Zr]Zr-DFO-, [89Zr]Zr-3,4,3-(LI-1,2-HOPO)- and [89Zr]Zr-DFO*-Cetuximab for Tumor Imaging: Which Chelator Is the Most Suitable?

“…To be able to directly compare the obtained in vivo pharmacokinetic data of [ 89 Zr]Zr-DFO*-cetuximab and [ 89 Zr]Zr-3,4,3-(LI-1,2-HOPO)-cetuximab, the chelating agents/complexes have to be introduced into the antibody using the same bioconjugation chemistry. Besides the number of derivatization sites per antibody molecule [ 18 , 19 , 20 ], also the type of chemical ligation can influence the obtained results [ 7 ]. In addition, [ 89 Zr]Zr-DFO-cetuximab had to be synthesized under identical conditions, enabling a direct comparison of both promising alternatives (DFO* and 3,4,3-(LI-1,2-HOPO)) with the clinically applied standard chelating agent for 89 Zr-labeling, DFO.…”

“…Ideally, the introduction of the chelator should be highly efficient under mild conditions, fully biocompatible, chemoselective and biorthogonal to enable efficient derivatization of antibody molecules. Further, it would be ideal to use a ligation technique that enables direct quantification of the number of introduced derivatization sites per antibody molecule as the latter determines the achievable immunoreactivity as well as the biodistribution of the bioconjugates [ 18 , 19 , 20 ]. One possibility to determine the number of chelators per antibody is by cumbersome isotopic dilution titration [ 21 ].…”

“…To address this issue, we developed a method for click chemistry-based chelator-antibody-modification relying on the inverse electron demand Diels-Alder (iEDDA) conjugation reaction between tetrazines and strained TCO (transcyclooctene) systems. This type of click chemistry reaction is very versatile and thus has found widespread use in radiochemistry [ 20 , 22 , 23 ]. In particular, this approach very recently showed that, in addition to conventional, very efficient antibody modification and subsequent direct one-step radiolabeling, it is also suitable for labeling antibodies in vivo via pretargeting [ 20 ].…”

“…This type of click chemistry reaction is very versatile and thus has found widespread use in radiochemistry [ 20 , 22 , 23 ]. In particular, this approach very recently showed that, in addition to conventional, very efficient antibody modification and subsequent direct one-step radiolabeling, it is also suitable for labeling antibodies in vivo via pretargeting [ 20 ].…”

The Inverse Electron Demand Diels‐Alder Reaction Between Tetrazine and Trans‐Cyclooctene for Pretargeted Bioimaging Applications

“…Notably, the IEDDA reaction between tetrazine (Tz) and trans ‐cyclooctene (TCO) is reported as one of the fastest bioorthogonal click reactions [18] . The second‐order reaction rate constant of the Tz−TCO click reaction can exceed 10 5 M −1 s −1 [20] . Thanks to its rapid kinetics and specific reactivity, the Tz−TCO click reaction shows great bioimaging potential even at low concentrations in complex biological contexts [21] .…”